Genetically modified mice and engraftment

ABSTRACT

A mouse with a humanization of the mIL-3 gene and the mGM-CSF gene, a knockout of a mRAG gene, and a knockout of a mII2rg subunit gene; and optionally a humanization of the TPO gene is described. A RAG/II2rg KO/hTPO knock-in mouse is described. A mouse engrafted with human hematopoietic stem cells (HSCs) that maintains a human immune cell (HIC) population derived from the HSCs and that is infectable by a human pathogen, e.g.,  S. typhi  or  M. tuberculosis  is described. A mouse that models a human pathogen infection that is poorly modeled in mice is described, e.g., a mouse that models a human mycobacterial infection, wherein the mouse develops one or more granulomas comprising human immune cells. A mouse that comprises a human hematopoietic malignancy that originates from an early human hematopoietic cells is described, e.g., a myeloid leukemia or a myeloproliferative neoplasia.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.12/897,517, filed Oct. 4, 2010; which claims the benefit of U.S.Provisional Application Nos. 61/249,069, filed Oct. 6, 2009; 61/256,237,filed Oct. 29, 2009; and 61/320,132, filed Apr. 1, 2010, whichapplications are incorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made with government support under AI070949 andAI079022 awarded by National Institute of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

This invention relates to the field of genetically modified non-humananimals, in particular immune-compromised mice having a RAG geneknockout, an II2rgII2rg gene knockout and a humanization of an IL-3 anda GM-CSF gene, and optionally a humanization of a TPO gene;RAG/II2rgII2rg knockout mice having a humanization of a TPO gene;genetically modified mice that are engrafted with human hematopoieticcells; and engrafted mice that are infected with a human pathogen, e.g.,Salmonella typhi or Mycobacterium tuberculosis.

BACKGROUND

Genetically modified mice, modified and engrafted mice, and their use inmodeling human diseases, e.g., for the purpose of drug testing, areknown in the art. Attempts have been made to use genetically modifiedmice to model a human immune system. A review of that field is providedin Manz (2007) Human-Hemato-Lympoid-System Mice: Opportunities andChallenges, Immunity, 26:537-541 hereby incorporated by reference.

To date no genetically modified mice have been generated thatdemonstrate infectivity with certain human pathogens, e.g., Salmonellatyphi (S. typhi). Even for pathogenic infections for which mouse modelsexist, the models can fail to adequately model certain pathologies inhumans, e.g., failure to form well-defined granulomas or granulomascontaining human immune cells in mouse models of Mycobacteriumtuberculosis (M. tuberculosis). In order to study the effects of certainpathogens on humans, and to test drugs for effectiveness in treatinghumans infected with certain pathogens, it would be useful to have anon-human animal such as a mouse that is genetically modified so that itis susceptible to infection with such a pathogen, e.g., S. typhi, and/orthat the infection more closely models human pathology, e.g., moreclosely models a human infection of M. tuberculosis.

In general, there is a need for genetically modified mice that cansupport maintenance and propagation of human hematopoietic stem cells,and for mice suitable for engraftment that can model or approximatecertain aspects of a human hemato-lymphoid system, e.g., in response toa human pathogen.

SUMMARY

Genetically modified non-human animals are provided. The non-humananimals include mice that comprise one or more knockouts of endogenousgenes and one or more humanized genes (i.e., replacement of anendogenous gene at its endogenous locus with a human ortholog orhomolog).

Genetically modified mice with ablated or compromised immune systems areprovided (e.g., via irradiation), as well as mice engrafted with humanhematopoietic cells or human hematopoietic stem and progenitor cells(HSPC). Genetically modified mice that comprise a human cell derivedfrom a human hematopoietic cell or HSPC are provided, as are mice thatcomprise a human hemato-lymphoid system.

Genetically modified, irradiated, and engrafted mice are provided thatare infectable with a human pathogen that does not infect wild-typemice. Mice are provided that in response to a human pathogen exposure(e.g., M. tuberculosis) mount an immune response having characteristics(e.g., formation of well-defined granulomas, or granulomas comprisinghuman immune cells) that are not observed in wild-type mice.

Genetically modified, irradiated, and engrafted mice for identifyingdrug-resistant strains of human pathogens, for testing human vaccines,and for developing and testing anti-pathogen drugs are provided, as wellas compositions and methods for using them.

Genetically modified mice capable of receiving and propagating humanimmune cells are provided, including mice that can sustain a humanhematopoietic malignancy.

In one aspect, a genetically modified mouse is provided, comprising: (a)a mouse RAG gene knockout; (b) a mouse II2rgII2rg gene knockout; and,(c) a humanization of one or more mouse genes selected from (i) a mouseIL-3 (mIL-3) gene, (ii) a mouse GM-CSF (mGM-CSF) gene, and (iii) a mousethrombopoietin (mTPO) gene.

In one embodiment, the RAG gene knockout is a RAG2 gene knockout.

In one embodiment, the humanization comprises replacement of a mTPO genewith a hTPO gene. In a specific embodiment, the humanization consistsessentially of humanization of a mTPO gene with a hTPO gene.

In one embodiment, the humanization comprises replacement of a mIL-3gene with a human IL-3 (hIL-3) gene and replacement of a mGM-CSF genewith a human GM-CSF (hGM-CSF) gene. In another embodiment, the mousefurther comprises replacement of a mTPO gene with a human TPO (hTPO)gene. In a specific embodiment, the humanization consists essentially ofhumanization of a mIL-3 gene with a hIL-3 gene and humanization of amGM-CSF gene with a hGM-CSF gene.

In one embodiment, the humanization comprises a replacement of a mGM-CSFgene with a human GM-CSF gene, and in the mouse human GM-CSF is notpredominantly expressed in liver and circulation. In one embodiment,human GM-CSF is predominantly expressed in the mouse lung. In oneembodiment, human GM-CSF expression is tissue-specific and reflectstissue specific expression in a human.

In one embodiment, the genetically modified mouse is treated so as toeliminate endogenous hematopoietic cells that may exist in the mouse. Inone embodiment, the treatment comprises irradiating the geneticallymodified mouse. In a specific embodiment, newborn genetically modifiedmouse pups are irradated sublethally. In a specific embodiment, newbornpups are irradiated 2×200 cGy with a four hour interval.

In one embodiment, the genetically modified and treated mouse isengrafted with human hematopoietic cells or human hematopoietic stemcells (HPSCs) to form a genetically modified and engrafted mouse. In oneembodiment, the hematopoietic cells are selected from human umbilicalcord blood cells and human fetal liver cells. In one embodiment,engraftment is with about 1-2×10⁵ human CD34+ cells.

In one embodiment, the genetically modified and engrafted mouse givesrise to a human cell selected from a CD34+ cell, a hematopoietic stemcell, a hematopoeitic cell, a myeloid precursor cell, a myeloid cell, adendritic cell, a monocyte, a granulocyte, a neutrophil, a mast cell, athymocyte, a T cell, a B cell, a platelet, and a combination thereof. Inone embodiment, the human cell is present at 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 monthsafter engraftment.

In one embodiment, the genetically modified and engrafted mouse givesrise to a human hemato-lymphoid system that comprises humanhematopoietic stem and progenitor cells, human myeloid progenitor cells,human myeloid cells, human dendritic cells, human monocytes, humangranulocytes, human neutrophils, human mast cells, human thymocytes,human T cells, human B cells, and human platelets. In one embodiment,the human hemato-lymphoid system is present at 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 monthsafter engraftment.

In one embodiment, the genetically modified and engrafted mouse exhibitsan inflammatory response mediated by a human cell. In a specificembodiment, the human cell is a macrophage. In a specific embodiment,the macrophage-mediated inflammatory response is mediated by an alveolarmacrophage. In a specific embodiment, the response mediated by thealveolar macrophage comprises a granuloma formation. In a specificembodiment, the granuloma comprises a human immune cell. In a specificembodiment, the granuloma is a well-organized granuloma. In a specificembodiment, the granuloma forms following exposure to a mycobacterium,e.g., M. tuberculosis. In one embodiment, the mouse exhibits aninflammatory response that comprises two or more granulomas. In oneembodiment, the genetically modified and engrafted mouse is a model forhuman M. tuberculosis infection.

In one embodiment, the genetically modified and engrafted mousecomprises an M. tuberculosis infection characterized at least in part bythe formation of a granuloma that comprises a human immune cell. In aspecific embodiment, the granuloma is a well-organized granuloma. In aspecific embodiment, the M. tuberculosis is a drug-resistant ormultidrug-resistant strain of M. tuberculosis that infects a humanpopulation. In one embodiment, the mouse infected with M. tuberculosiscomprises a granuloma in a lung. In a specific embodiment, the granulomais a well-developed granuloma. In a specific embodiment, the granulomain the lung comprises human immune cells. In a specific embodiment thehuman immune cells of the granuloma are selected from an activated humanmacrophage, an activated human T cell, and a combination thereof.

In one embodiment, the genetically modified and engrafted mouse exhibitsenhanced mucosal immunity as compared with an engrafted mouse that lacksa humanization of one or more of IL-3, GM-CSF, and TPO genes. In aspecific embodiment, the enhanced mucosal immunity comprises an enhancedexpression of interferon β (IFNβ) following influenza A infection.

In one embodiment, the genetically modified and engrafted mousecomprises an infection selected from a M. tuberculosis and a S. typhiinfection. In one embodiment, the mouse reproduces S. typhi or M.tuberculosis. In one embodiment, the mouse mounts an anti-mycobacterialimmune response to a human pathogenic mycobacterium, wherein theresponse comprises formation of a granuloma mediated by human immunecells and that comprises a human immune cell. In a specific embodiment,the granuloma is a well-developed granuloma.

In one embodiment, the genetically modified and engrafted mousecomprises a humanization that comprises humanization of a mTPO gene toform a hTPO engrafted mouse. In one embodiment, the hTPO engrafted mouseexhibits an increase of human meyloid cells in bone marrow over anengrafted mouse that comprises a mTPO gene but no hTPO gene. In aspecific embodiment, the human myeloid cells are increased 1.5-fold,2-fold, 2.5-fold, or 3-fold over an engrafted mouse that lacks a hTPOgene. In a specific embodiment, the increase in granulocytes is about1.5-fold, 2-fold, 2.5-fold, or 3-fold. In another embodiment, anincrease in peripheral blood monocytes is observed over an engraftedmouse that lacks a hTPO gene, wherein the increase in peripheral bloodmonocytes is about 1.5-fold, 2-fold, 2.5-fold, or 3-fold. In oneembodiment, the genetically modified engrafted mouse comprises ahumanization that consists essentially of a hTPO gene that replaces amTPO gene, wherein the mouse does not express a mouse TPO but expressesa human TPO.

In one aspect, a genetically modified and engrafted mouse is provided,comprising a knockout of a Rag gene, an II2rgII2rg knockout, and ahumanization of TPO, wherein the mouse is engrafted with humanhematopoietic stem cells, or human immune cells, and comprises a humanhematopoietic malignancy that originates from an early humanhematopoietic cell. In a specific embodiment, the malignancy is selectedfrom a myeloid leukemia and a myeloproliferative neoplasia.

In one embodiment, the mouse further comprises a human IL-3 gene and ahuman GM-CSF gene, and a knockout of an endogenous mouse IL-3 gene and aknockout of an endogenous mouse GM-CSF gene.

In one aspect, a mouse is provided that comprises a RAG gene knockout,an II2rg gene knockout, and a genetic modification that provides humanmyeloid cells with a competitive advantage with respect to mouse myeloidcells. In one embodiment, the genetic modification is a replacement of amouse gene required for mouse myeloid cell development and/ormaintenance with a counterpart human gene. In one embodiment, thegenetic modification is selected from a replacement of a mouse IL-3 genewith a human IL-3 gene, replacement of a mouse GM-CSF gene with a humanGM-CSF gene, and a combination thereof. In one embodiment, the mouselacks or substantially lacks endogenous mouse hematopoietic cells andcomprises human hematopoietic cells.

In one aspect, a method for making a mouse that is infectable with ahuman pathogen is provided, comprising genetically modifying andengrafting a mouse as described herein and exposing the geneticallymodified and engrafted mouse to a human pathogen, and maintaining themouse under conditions sufficient for the human pathogen to infect themouse. In one embodiment, the human pathogen is selected from M.tuberculosis and S. typhi. In one embodiment, the human pathogen is ahuman pathogen that is not pathogenic in a mouse that lacks the geneticmodification(s). In one embodiment, the human pathogen is a humanpathogen that does not infect a mouse that lacks the geneticmodification(s).

In one aspect, a method for determining the effect of a drug on a humanpathogen is provided, comprising exposing a genetically modified andengrafted mouse as described herein to a human pathogen, allowing thepathogen to infect the mouse, and measuring a parameter of the infectionover time in the presence and in the absence of the drug. In oneembodiment, the human pathogen is a pathogen that does not infect amouse that lacks the genetic modification(s). In one embodiment, thehuman pathogen is selected from M. tuberculosis and S. typhi. In oneembodiment, the mouse is exposed to a known number of infectious unitsof the human pathogen, and the parameter of infection is the number ofinfectious units of the human pathogen in a fluid or tissue of themouse.

In one embodiment, the parameter of the infection is a titer in a bodyfluid of the mouse. In one embodiment, the infection is selected from anM. tuberculosis infection and a S. typhi infection. In a specificembodiment, the infection is an M. tuberculosis infection and theparameter is formation of a granuloma. In a specific embodiment, thegranuloma is a lung granuloma. In another specific embodiment, thegranuloma is a well-defined granuloma.

In one aspect, a genetically modified mouse is provided, comprising: (a)a mouse RAG gene knockout; (b) a mouse II2rg gene knockout; and, (c) ahumanization of (i) a mouse IL-3 (mIL-3) gene, and a (ii) a mouse GM-CSF(mGM-CSF) gene; wherein the mouse following irradiation to ablateendogenous mouse hematopoietic cells and following engraftment withhuman hematopoietic stem cells maintains the human hematopoietic stemcells and develops from the human hematopoietic stem cells a humanimmune cell population that comprises functional differentiated humanimmune cells that include human myeloid progenitor cells, human myeloidcells, human dendritic cells, human monocytes, human granulocytes, humanneutrophils, human mast cells, human thymocytes, human T cells, human Bcells, and human platelets. In another aspect the mouse furthercomprises (iii) a humanization of a mouse thrombopoietin (mTPO) gene.

In one embodiment, the mouse maintains a population of human immunecells that is as diverse in cell type as the population of immune cellsin a human. In one embodiment, the human immune cells are maintained forat least at 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,10 months, 11 months, or 12 months after engraftment.

In one embodiment, the mouse upon exposure to a human pathogen orantigen of a human pathogen mounts a cellular and/or humoral immuneresponse that models infection of a human exposed to the pathogen. Inone embodiment, the human pathogen is a pathogen that does not infect awild-type mouse. In another embodiment, the human pathogen is a pathogenthat infects a wild-type mouse, wherein the wild-type mouse followinginfection does not model an immune response that a human mounts inresponse to the pathogen. In one embodiment, the pathogen is a virus, amycobacterium, a fungus, or a bacterium. In specific embodiments, thepathogen is a human or porcine or avian influenza virus, S. typhi, or M.tuberculosis.

Further applications and embodiments of the invention will becomeapparent to those skilled in the art upon reading this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows S. typhi infection in engrafted RAG KO, II2rg KO and RAGKO, II2rg KO, hIL-3/hGM-CSF mice ten days post-infection. Experimentalgroups: (1) Engrafted: n=9 (4 m/m, 5 h/m); engraftment inblood=6.5-16.7%; (2) Control: n=8; engraftment in blood=0.04-0.4%(reflects flow cytometry background; Control mice were unengrafted).

FIG. 2 shows S. typhi infection in spleens of engrafted RAG KO, II2rg KOmice a week post-infection with 1×10³ S. typhi.

FIG. 3 shows S. typhi infection in spleens and livers of engrafted RAGKO, II2rg KO mice 4 weeks post-infection with 1×10⁴ S. typhi, whereinthe mice were engrafted with CD34-positive fetal liver cells.

FIG. 4 shows S. typhi infection in gall bladders of RAG KO, II2rg KOmice 4 weeks post-infection with 1×10⁴ S. typhi, wherein the mice wereengrafted with CD34-positive fetal liver cells.

FIG. 5 (a)-(d) shows results of validation studies of hGM-CSF expressionin non-engrafted hIL-3/hGM-CSF mice.

FIG. 5(e) shows a humanization strategy at a mouse IL-3/GM-CSF locus.

FIG. 6(a)-(e) shows results of lung studies of engrafted humanized(hIL-3/hGM-CSF) mice.

FIG. 6(f),(g) shows ELISA results for mouse and human IL-3 (f) andGM-CSF (g) production by activated splenocytes.

FIG. 7(a) shows PAS staining of lung tissue sections from non-engraftedor engrafted m/m or h/h KI mice; (b) quantification of total protein inBAL fluid from non-engrafted (non) or engrafted h/h KI mice or m/mcontrol mice (n=6 per group).

FIG. 8(a) shows expression of human Hprt normalized to mouse Hprt; (b)expression of human IFNγ normalized to mouse Hprt; (c) expression ofhuman IFNγ normalized to human Hprt

FIG. 8(d) shows flow cytometry analysis of human bone marrow cells fromengrafted hIL-3/GM-CSF h/m KI mice in steady state; (e) flow cytometryanalysis of human blood cells from CB-engrafted m/m or h/m KI mice 72 hafter two i.p. injections of LPS; (f) frequency of human CD14+ bloodcells in engrafted m/m or h/m KI mice 72 h post-LPS injections; (g)ELISA of human IL-6 in sera from engrafted m/m or h/m KI mice 2-3 hafter first (top) and second (bottom) LPS injection.

FIG. 9(a) shows frequency of human T cells (hCD45+hCD3+) in the lung;(b) distribution of human CD4 and CD8 T cells in the lung; (c) ratio ofhuman CD4 to CD8 T cells in lung; (d) flow cytometry analysis ofsplenocytes from BALB/c mice, engrafted m/m mice, and engrafted h/m KImice four weeks after BCG infection; (e) quantitative RT-PCR analysis ofhuman IFNγ (left) and TNFα (right) gene expression in lung tissue fromBALB/c mice, non-engrafted (non) m/m mice, engrafted m/m mice, andengrafted h/m KI mice four weeks after BCG infection.

FIG. 9(f) shows DiffQuick™ staining of BAL cells from non-engrafted m/mor h/h KI mice; magnification 400×; (g) PAS staining of lung tissuesections from non-engrafted m/m or h/h KI mice; magnification 400×.

FIG. 10(a) shows hematoxylin and eosin (H&E) staining of lung tissuesections from engrafted h/m KI mice four weeks after BCG infection;magnification 100× (left) and 200× (right); (b) lung tissue sectionsstained for human CD45, CD3, or CD68 from engrafted h/m KI mice fourweeks after BCG infection; magnification 200×.

FIG. 11(a) shows RT-PCR analysis of mouse TPO (mTpo) and human TPO(hTPO) expression in different tissues of a Rag2^(+/−)γ_(c) ^(Y/−)TPO^(h/m) mouse; (b) RT-PCR analysis of mTpo and hTPO expression inliver, kidney and mesenchymal multipotent stromal cells (MSCs) ofRag2^(−/−)γ_(c) ^(−/−) TPO^(m/m), TPO^(h/m) and TPO^(h/h) mice; (c)concentrations of mouse and human TPO proteins measured by ELISA inserum of TPO^(m/m), TPO^(h/m) and TPO^(h/h) mice.

FIG. 11(d) shows a targeting construct for replacing mTPO gene with hTPOgene.

FIG. 12(a) shows FACS analysis of human and mouse CD45⁺ cells in bonemarrow of Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) mice 3 to 4months after engraftment with human CD34⁺ cells; (b) percentages ofhuman CD45⁺ cells in the bone marrow 3 to 4 months (left) or 6 to 7months (right) after transplantation; (c) absolute numbers of humanCD45⁺ cells in the bone marrow of the same animals as in (b).

FIG. 12(d) shows the percentages of human CD45⁺ cells in the bone marrowof Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) mice, engrafted withhuman CD34⁺ cells isolated from cord blood (CB) or fetal liver (FL).

FIG. 13(a) shows platelet counts in the blood of adult non-engraftedRag2^(−/−)γ_(c) ^(−/−) TPO^(m/m), TPO^(h/m) and TPO^(h/h) mice; (b)representative FACS analysis of mouse (mCD61⁺) and human (hCD41a⁺)platelets in the blood of Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h)mice 3 to 4 months after engraftment; (c) human platelet chimerism,determined by FACS, in TPO^(m/m) and TPO^(h/h) mice; (d),(e) counts ofmouse (mCD61⁺, 20d) and human (hCD41a⁺, 20e) platelets in the blood ofTPO^(m/m) and TPO^(h/h) recipients; (f) human megakaryocyte percentages(CD41a⁺) among human CD45⁺ cells in the bone marrow.

FIG. 13(g),(h) shows percentages of human CD45⁺ cells in blood andspleen of Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) mice; (i)provides total cellularity of the thymi of engrafted TPO^(m/m) andTPO^(h/h) recipients.

FIG. 14(a) shows FACS analysis of human myeloid cell populations in bonemarrow and blood of Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) mice3 to 4 months after engraftment; (b) total myeloid populations (CD33+cells); (c) granulocytes (CD33⁺CD66^(hi)); (d) DiffQuick™ staining ofhCD45⁺SSC^(hi)CD33⁺CD66^(hi) cells purified from the bone marrow ofTPO^(h/h) recipients; (e) monocytes (CD33⁺CD66^(lo)CD14⁺); (f),(g)analysis of human myeloid cell populations relative to total human CD45⁺cell chimersim in blood of Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) andTPO^(h/h) recipients; (f) granulocytes (CD66⁺); (g) monocytes (CD14⁺).

FIG. 15(a) shows FACS analysis of mouse Lin⁻ Sca1+ c-Kit⁺ stem andprogenitor cells in the bone marrow of non-engrafted Rag2^(−/−)γ_(c)^(−/−) TPO^(h/m) and TPO^(h/h) mice compared to WT TPO (TPO^(m/m))Rag2^(−/−)γ_(c) ^(−/−) mice; (b) quantitative analysis of the resultspresented in (a); (c) FACS analysis of human CD34⁺CD38⁻ cells in thebone marrow of Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) mice 3 to4 months after engraftment; (d) quantitative analysis of the percentagesof CD38⁻ cells in the human CD45⁺CD34⁺ population in TPO^(m/m) andTPO^(h/h) recipient mice; (e) human CD34⁺CD38⁻ cells in the bone marrowof the same mice as in 15(d); (f),(g) methylcellulose colony formationassay with human CD45⁺CD34⁺ cells purified from Rag2^(−/−)γ_(c) ^(−/−)TPO^(m/m) and TPO^(h/h) recipients; (f) is CFU-GEMM, (g) is BFU-E(black), CFU-G (white), CFU-M (gray) and CFU-GM (dashed); (h) humanCD45+ chimerism in secondary transplant of human CD45⁺CD34⁺ cells fromRag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) mice into newbornRag2^(−/−)γ_(c) ^(−/−) mice.

DETAILED DESCRIPTION

The invention is not limited to particular embodiments discussed, but isdescribed by the granted claims.

Unless otherwise specified, all technical and scientific terms usedherein include the same meaning as commonly understood by one ofordinary skill in the art to which the invention belongs. Although anymethods and materials similar or equivalent to those described can beused in making or using the invention, particular embodiments, methods,and materials are now described. All publications mentioned are herebyincorporated by reference. The present disclosure supersedes anydisclosure of an incorporated publication to the extent that acontradiction exists.

The singular forms “a”, “an”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a gene” includes a plurality of such genes and reference to “the geneknockout” includes reference to one or more knockouts and equivalentsthereof.

Modified Mice that Support Human Immune Cells: hIL-3/GM-CSF Mice

Mice with components of the human immune system (HIS mice) hold greatpromise for studying the human immune system in vivo and for testinghuman vaccines and testing and developing drugs to treat human diseasesand disorders. HIS mice are generated by transplanting a severelyimmunodeficient mouse strain (such as recombination-activating gene 2(Rag2) knockout (KO) interleukin 2 receptor gamma (II2rg) KO mice) withhuman hematopoietic stem and progenitor cells. Compared to nonhumanprimates, HIS mice have the advantages of a small animal model, i.e.,they allow more versatile experimentation, are more accessible to theresearch community, and are ethically more acceptable than conductingexperiments with human subjects. Most importantly, experimental findingsderived from HIS mice might be more relevant and applicable to humans,because infection with human-specific pathogens and the study ofhuman-specific immune responses and immunopathologies are now becomingfeasible.

Although much progress has been made in recent years, current HIS micemodels have several major limitations such as the poor development,maintenance, and function of human myeloid and T cells. As aconsequence, human inflammatory and immune responses at mucosal surfacesor robust human T cell-mediated responses, such as delayed-typehypersensitivity (DTH), have rarely been observed. Thus, current HISmice are not well suited to study infection and pathology caused by theserious human pathogen Mycobacterium tuberculosis. Indeed,granulomas—specifically granulomas containing human cells, a hallmark ofthe human immune response to mycobacteria—have so far not been reportedin HIS mice (see, e.g., Manz et al. (2009) Renaissance for mouse modelsof human hematopoiesis and immunobiology, Nat. Immunol. 10:1039-1042).

Current HIS mouse hosts are not well suited to model certain infections,at least in part because current HIS mouse hosts present anon-physiological environment for human cells. Several mouse cytokines,e.g., IL-3 and GM-CSF, do not act on the human cognate receptors. Inaddition, Rag2 KO II2rg KO mice have an intact mouse myeloidcompartment, and human myeloid cells might have a competitivedisadvantage relative to host cells. To overcome these limitations, thisdisclosure describes generating human cytokine knock-in mice where genesencoding mouse cytokines are replaced by their human counterparts.Criteria for cytokine replacement are: (1) the mouse cytokine does notor weakly act on human cells; (2) the human cytokine does not or weaklyact on mouse cells to confer competitive advantage to human cells; (3)the human cytokine is not exclusively produced by hematopoietic(transplanted) cells; (4) lack of the mouse cytokine is not lethal tomouse host, or the human KI cytokine is sufficiently cross-reactive torescue the mouse KO phenotype. The KI strategy should allow faithfulexpression in appropriate organs and at physiologic concentrations.Importantly, in homozygous KI mice, human cognate receptor expressingcells should gain a competitive advantage over respective mouse cells.

IL-3 and GM-CSF are two cytokines crucial for myeloid cell developmentand function. Neither cytokine is cross-reactive between human andmouse. IL-3 stimulates early hematopoietic progenitors in vitro, but isdispensable for steady-state hematopoiesis in vivo. However, togetherwith GM-CSF it is required for effective DTH responses in vivo. IL-3also specifically stimulates the proliferation of alveolar macrophages(AM) in vitro. GM-CSF is highly expressed in the lung and important forlung homeostasis in vivo, as demonstrated by the fact that GM-CSF KOmice develop pulmonary alveolar proteinosis (PAP) which is characterizedby protein accumulation in the lung due to defective surfactantclearance. Alveolar macrophages from GM-CSF KO mice have a defect interminal differentiation, which leads to impaired innate immunity topathogens in the lung. GM-CSF also stimulates the proliferation of humanAM in vitro. Similar to IL-3, GM-CSF is largely dispensable forsteady-state hematopoiesis, and the same applies to mice lacking bothcytokines. In contrast, GM-CSF is required for inflammatory responsessuch as the production of pro-inflammatory cytokines by macrophages andthe mobilization and recruitment of monocytes. GM-CSF is also essentialfor protective immunity against a range of pathogens, including M.tuberculosis. In particular, GM-CSF KO mice infected with M.tuberculosis do not develop granulomas, a hallmark of tuberculosis.

This disclosure is based at least in part on the realization thatgenerating hIL-3/GM-CSF KI mice would be valuable to support humanmyeloid cell reconstitution and function as well as human innate immuneresponses to pathogens in mice. Results obtained and described in thisdisclosure with such KI mice demonstrate that this strategy affords asubstantial improvement over current models of HIS mice in terms ofhuman myeloid cell development, human lung mucosal immunity, and alsogranuloma formation after mycobacterial infection. These and otherbeneficial properties of such mice are discussed elsewhere in thisdisclosure.

The ability to study human tissue in an in vivo setting in mice hasopened a wide range of possible avenues of research. Major limitationshave hindered the application of the approach and of these one of themost important deficiencies has been the inability of mouse factors tosupport human cells. Indeed, in the immune system, many essentialfactors required for human immune cell development and function arespecies-specific and cannot be effectively provided by the mouse. It wastherefore decided to follow a strategy of replacing the mouse genes withtheir human counterparts, enabling the better development and functionof human cells and potentially disabling the same of the correspondingmouse cells. By applying this concept to human cytokine KI mice, proofof concept is provided here that replacement of immune genes in themouse host with human genes improves HIS mice. Specifically, thisdisclosure supports the notion that inappropriate cytokinecrossreactivity between mouse and human, and having to compete withmouse cells, indeed limit engraftment and function of human myeloidcells in current HIS mice.

Human cytokines can be delivered to HIS mice by intravenous injection,e.g., to boost human NK cell and T cell reconstitution by injections ofIL-15/IL-15Rα complexes and IL-7, respectively. Another approach is thehydrodynamic injection of plasmid DNA expressing human cytokines, whichleads to transient expression in the liver. This approach has veryrecently been used to improve reconstitution of human DC by hydrodynamicdelivery of GM-CSF and IL-4 (see, Chen et al. (2009) Expression of humancytokines dramatically improves reconstitution of specific human-bloodlineage cells in humanized mice, Proc Natl Acad Sci USA,106(51):21783-21788). In contrast to the present disclosure, nofunctional responses of myeloid cells or in vivo responses to pathogenswere reported in these mice. Finally, human cytokines can also beoverexpressed as transgenes in HIS mice. This approach has been used togenerate human IL-3/GM-CSF/stem cell factor (SCF) transgenic (tg) mice(see, Nicolini et al. (2004) NOD/SCID mice engineered to express humanIL-3, GM-CSF and Steel factor constitutively mobilize engrafted humanprogenitors and compromise human stem cell regeneration, Leukemia18:341-347). In these mice human cytokine expression is driven by thecytomegalovirus promoter, which leads to ubiquitous expression. However,hIL-3/GM-CSF/SCF tg HIS mice are hampered by reduced maintenance ofhuman hematopoietic stem cells in bone marrow and expanded terminalmyelopoiesis. Again, unlike the present disclosure, improved myeloidcell function or in vivo responses were not described. By contrast, inthe system described here, physiologic expression of the targeted genesin steady state and inflammation enables appropriate development andfunction of the appropriate cell type only. Importantly, the approachdescribed in this disclosure generates strains of mice that can bemaintained and propagated under highly reproducible conditions and madeavailable worldwide for studies.

The hIL-3/GM-CSF KI mice described in this disclosure represent aconsiderable improvement over previous HIS mice and the alternativeapproaches discussed above. First, delivery of human IL-3 and GM-CSF bythe KI strategy described here leads to long-term cytokine expression,which circumvents the need for repeated injections of expensivecytokines. Second, faithful expression in organs where IL-3 and GM-CSFare normally expressed is achieved. Under physiological conditions,GM-CSF is mainly expressed in the lung (FIG. 5a ). In contrast,hydrodynamic delivery leads to predominant expression in the liver andin the circulation. In both organs GM-CSF is not expressed insteady-state conditions. Third, physiological amounts of IL-3 and GM-CSFare expressed in KI mice in contrast to delivery by hydrodynamicinjection or ubiquitous overexpression in hIL-3/GM-CSF/SCF tg mice. Ithas been demonstrated that physiological levels of GM-CSF are importantfor a protective immune response against M. tuberculosis. Thus,transgenic mice with local overexpression of GM-CSF in the lung showdefective granuloma formation and increased susceptibility to M.tuberculosis. Similarly, intravenous administration of GM-CSF also leadsto impaired control of M. tuberculosis infection in mice. Fourth,homozygous hIL-3/GM-CSF KI mice allow the simultaneous impairment of themouse myeloid compartment since mouse IL-3 and GM-CSF are not expressedin homozygous mice. This leads to a competitive advantage for humanmyeloid cells as shown in the present disclosure.

Tuberculosis caused by infection with M. tuberculosis results in 1.7million deaths per year. Therefore, novel effective preventive andtherapeutic measures are urgently needed. While mice can be infectedwith M. tuberculosis, they do not represent an ideal model for humantuberculosis. This is due to species-specific differences in the immuneresponse to M. tuberculosis. For example, infected mice do not developwell-organized granulomas. Granulomas are the hallmark of the immuneresponse in humans with tuberculosis and contain activated macrophagesthat fuse to form epitheloid and multinucleated giant cells, andactivated T cells. Granulomas play an important role in limitingbacterial replication and in controlling spread of mycobacteria. GM-CSFpromotes the differentiation of AM into multinucleated giant cells invitro. Studies in transgenic mice also revealed a role for GM-CSF in thefusion of macrophages to form multinucleated giant cells in vivo.Furthermore, GM-CSF is essential for granuloma formation aftermycobacterial infection. Absence of granulomas in GM-CSF KO miceinfected with M. tuberculosis is associated with increased bacterialreplication and reduced survival. Finally, humans with PAP, caused bydefective GM-CSF signaling, show increased susceptibility tomycobacterial infections.

Human anti-mycobacterial immune responses, particularly formation ofgranulomas by human cells, have not been previously reported in HISmice. This is likely due to weak human macrophage and T cell responses.In this disclosure, an antigen-specific T cell response to mycobacteriawas detected in a subset of mice engrafted with human cells. Inaddition, given the prominent role of GM-CSF in granuloma biology, it ishypothesized that engrafted hIL-3/GM-CSF KI mice would be a better hostto support granuloma formation. This was indeed the case in at least asubset of mice infected with BCG. Importantly, lung granulomas in thesemice contained human T cells and human macrophages, although thegranulomas had the loose architecture typical of mouse granulomas.Future efforts should aim to further boost human T cell and macrophageresponses in HIS mice. This should lead to the development of a smallanimal model that allows the study of human immune responses to M.tuberculosis in vivo. hIL-3/GM-CSF KI mice could also be useful in othersettings to study the human immune response in vivo. This includesinfection with pulmonary pathogens, autoimmunity, and human cancers. Insummary, the hIL-3/GM-CSF KI mice presented in the current disclosurerepresent a considerable improved HIS mouse model that should serve as aversatile tool for future studies.

Modified Mice that Support Human Immune Cells: hTPO

Hematopoietic stem cells (HSCs) are characterized by two majorproperties: life-long self-renewal, and differentiation capacity to allmature hematopoietic lineage cells. To ensure HSC pool homeostasis, itis believed that upon cell division, HSCs generate one functional HSCwhile the other offspring cell might undergo a highly organized programof differentiation and cellular expansion, during which multiplelineages of committed progenitors, and ultimately terminallydifferentiated cells are produced.

Mouse hematopoiesis has been extensively studied during the pastdecades, leading to the identification and functional characterizationof immuno-phenotypically defined cellular populations, highly enrichedin stem and progenitor cells in vivo. However, prospective experimentalin vivo studies of human hematopoiesis have been limited by obviouspractical and ethical restrictions.

To circumvent this limitation, several xenogeneic transplantation modelsfor in vivo human hematopoiesis studies have been developed. Of these,transplantation of human hematopoietic cells into immunodeficient micehas been broadly established in experimental hematopoiesis laboratories.The models most commonly used today rely on the BALB/c Rag2^(−/−)γ_(c)^(−/−) or NOD-SCID γ_(c) ^(−/−) strains of mice. Both strains are highlyimmunodeficient, lacking B, T and NK cells, and their genetic backgroundis permissive for human hematopoietic engraftment and differentiation.Upon human CD34⁺ hematopoietic stem and progenitor cell transplantation,most human hematopoietic populations (including B cells, T cells,monocytes, dendritic cells, erythrocytes and platelets) can develop andare detectable in these models. However in those chimeric animals, thereis a bias towards lymphoid development with initially high B cell countsthat typically represent up to 80% of human cells, myelo-monocyticdevelopment is minor, and engraftment levels usually start to decline4-6 months after transplantation. Moreover, the xenogenic engraftment ofhuman cells into mice requires transplantation of large numbers of cellscompared to numbers sufficient for the optimal engraftment of mousehematopoietic stem and progenitor cells into mice, or human cells intohumans, respectively. Furthermore, in contrast to mouse HSCstransplanted into mouse recipients, human HSCs do not expand, nor arethey maintained, in the xenogeneic mouse environment. Thus the mousebackground does not provide an optimal environment to study thephysiology of human HSCs. This might be due to absence or limitedcross-reactivity of growth factors, required to support the function andmaintenance of HSCs.

Thrombopoietin (TPO) was initially identified as a growth factor thatpromotes the development of megakaryocytes and platelets. TPO isconstitutively produced by the liver and the kidneys and released intothe blood circulation. The receptor for TPO, c-Mpl, is expressed byhematopoietic stem and progenitor cells in the bone marrow. C-Mpl isalso expressed on circulating platelets. However, the binding of TPO onplatelets does not activate any signaling pathway. Thus, thrombocytesact as a sink or scavengers for TPO and via this mechanism contribute tonegative regulation of thrombopoiesis. Subsequently, TPO has beenrecognized for its important function to support the expansion andself-renewal of HSCs. TPO deficiency leads to reduced numbers of HSCs inadult mice, and the presence of TPO is needed to maintain adult HSCs inquiescence. Furthermore, TPO is required to support post-transplantationexpansion of HSCs, necessary to replenish the hematopoietic compartmentof irradiated hosts. Interestingly, it has been demonstrated thatosteoblastic cells involved in forming the HSC niche in the bone marrowproduce TPO, critical for HSC function and maintenance.

Although mouse and human TPO are both-sided cross-reactive to therespective cognate receptors when used at supraphysiological doses invitro, affinity and biologic activity might differ when the cytokineacts at limiting, physiological doses in context of an in vivoenvironment. Thus, mouse TPO might not provide an appropriate stimulusto the human c-Mpl receptor in vivo, and therefore could account for theimpaired properties of human HSCs in the mouse environment. To correctthis potential defect, the gene encoding mouse TPO was replaced by itshuman counterpart in Rag2^(−/−)γ_(c) ^(−/−) mice.

This disclosure is based at least in part on the realization thatgenerating hTPO KI mice in a RAG2^(−/−)γ_(c) ^(−/−) background would bevaluable to support human myeloid cell reconstitution and function aswell as human innate immune responses to pathogens in mice, in the micethemselves and in the progeny of such mice bred with hIL-3/GM-CSF mice.Results obtained from hTPO KI mice in a RAG2^(−/−)γ_(c) ^(−/−)background are described in this disclosure. Homozygous hTPO KI mice hadsignificantly increased levels of human engraftment in bone marrow, andmultilineage differentiation of hematopoietic cells was improved overmTPO mice, the hTPO KI mice displaying an increased ratio ofmyelomonocytic vs. lymphoid lineages. Both the number and self-renewalcapacity of human stem and progenitor cells were improved, asdemonstrated by serial transplantation. Thus, among other applications,hTPO KI mice are useful for propagation of human cells by serialtransplantation.

Breeding a hIL-3/GM-CSF Mouse and a hTPO Mouse

Progeny of hIL-3/GM-CSF and hTPO mice described herein are expected tohave at least the same relevant characteristics and display at least thesame benefits as parental lines (La, hIL-3/GM-CSF mice and hTPO mice).For example, human cell populations from either parental strain, orboth, or such a progeny can be isolated and serially transplanted intoeither a hTPO mouse or a progeny of an hIL-3/GM-CSF and hTPO mouse.Thus, in one aspect, a genetically modified mouse is provided that is aprogeny of a hIL-3/GM-CSF mouse and a hTPO mouse as described herein(including progeny that are bred to homozygosity with respect to eachrelevant gene) and wherein the genetically modified mouse exhibits thebenefits and characteristics of both a hIL-3/GM-CSF mouse and a hTPOmouse. In one aspect, such a progeny mouse is provided that comprises anablated immune system (e.g., an irradiated mouse), and is suitable forengraftment and/or serial transplantation from any engrafted mouse(e.g., an engrafted mouse as described herein).

Engrafting Genetically Modified Mice

A genetically modified mouse in accordance with the invention finds oneuse as a recipient of human hematopoietic cells that is capable ofdeveloping human immune cells from engrafted human hematopoietic cells.In one embodiment, human hematopoietic cells or human hematopoietic stemand progenitor cells (HSPC) are placed (engrafted) in a geneticallymodified and irradiated mouse in accordance with the invention. Thehuman hematopoietic cells or human hematopoietic stem cells give rise inthe genetically modified mouse to a cell selected from a humanCD34-positive cell, a human hematopoietic stem cell, a humanhematopoietic cell, a myeloid precursor cell, a myeloid cell, adendritic cell, a monocyte, a neutrophil, a mast cell, and a humanhemato-lymphoid system (including human hematopoietic stem andprogenitor cells, human myeloid progenitor cells, human myeloid cells,human dendritic cells, human monocytes, human granulocytes, humanneutrophils, human mast cells, human thymocytes, human T cells, human Bcells, human platelets), and a combination thereof.

The genetically modified mice can be irradiated to eliminate endogenoushematopoietic cells that may be present, and the mouse can be engraftedwith any suitable source of human hematopoietic cells. One suitablesource of hematopoietic cells known in the art is human umbilical cordblood cells, in particular CD34-positive cells. Another source ofhematopoietic cells is human fetal liver.

In one embodiment, engraftment of a mouse in accordance with theinvention with human hematopoietic cells results in a mouse thatexhibits an enhanced number of human hematopoietic cells than animmune-compromised mouse that lacks the humanization of a TPO gene,lacks humanization of a IL-3 and a GM-CSF gene, or lacks humanization ofa TPO gene and a IL-3 and a GM-CSF gene.

In one embodiment, engraftment of a mouse in accordance with theinvention with human hematopoietic cells results in a mouse thatexhibits an enhanced number of human blood cells (e.g., maturehematopoietic cells) as compared with an immune-compromised mouse thatlacks the humanization(s). In a specific embodiment, the humanhematopoietic cells are selected from human CD34-positive cells,hematopoietic stem cells, hematopoietic cells, myeloid precursor cells,myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils,mast cells, and a human hemato-lymphoid system (including humanhematopoietic stem and progenitor cells, human myeloid progenitor cells,human myeloid cells, human dendritic cells, human monocytes, humangranulocytes, human neutrophils, human mast cells, human thymocytes,human T cells, human B cells, human platelets), and a combinationthereof.

Nonlimiting Applications of Genetically Modified Engrafted Mice

A genetically modified mouse engrafted with human hematopoietic cells isa useful animal in which to study pathogens that do not normally infectmice. One such example is the causative agent of typhoid fever, S.typhi.

Typhoid fever afflicts over 21 million people around theworld—principally in the developing world—including about 400 cases/yearin the United States. Typhoid fever has been treated with the drugsamoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime,chloramphenicol, ciprofloxacin, co-trimoxazole, ertapenem, imipenem,fluoroquinolones (e.g., ciprofloxacin, gatifloxacin, ofloxacin),streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, andcombinations thereof. Recurrent infections are common, which limitsdisease management by antibiotic therapy. Further, multi-drug resistanceis also prevalent with S. typhi infections.

New therapeutics, new vaccines, and new ways of testing efficacy oftherapeutics and vaccines are needed. A mouse capable of being infectedby S. typhi, for example, would be useful to identify new therapeuticsand new vaccines. New therapeutics and new vaccines could be testing insuch a mouse by, e.g., determining the amount of S. typhi in the mouse(in blood or a given tissue) in response to treatment with a putativeanti-S. typhi agent, or by inoculating the mouse with a putative vaccinefollowed by exposure to an infective administration of S. typhi, andobserving any change in infectivity due to inoculation by the putativevaccine as compared to a control not inoculated with the vaccine butinfected with S. typhi.

A genetically modified and engrafted mouse in accordance with theinvention is useful to make a mouse that is infectable by a humanpathogen that does not infect mice. For example, the mouse is useful asa non-human animal infectable by S. typhi. In one embodiment, thegenetically modified and engrafted mouse displays an enhancedengraftment of human cells as compared to an engrafted mouse that lacksthe genetic modification(s), wherein the enhancement is sufficient tomaintain a S. typhi infection. In a specific embodiment, maintenance ofa S. typhi infection includes the ability of S. typhi to reproduce inthe mouse. In a specific embodiment, the S. typhi infection includes theability of the infected mouse to reproduce S. typhi. In a specificembodiment, the mouse is capable of reproducing S. typhi at least aweek, 10 days, two weeks, three weeks, or four weeks following aninitial introduction or infective exposure of S. typhi.

A method for identifying an anti-S. typhi agent, is also provided,wherein the method employs a mouse as described herein that isinfectable by S. typhi. Wild-type mice, and other knownimmune-compromised mice (e.g., RAG1/RAG2 gene knockout mice) are notcapable of being infected by S. typhi.

A genetically modified mouse comprising an II2rg gene knockout and a RAGgene knockout (e.g., a RAG 2 gene knockout) (first type) and alsocomprising a replacement of the endogenous mouse IL-3 gene with a humanIL-3 gene and the endogenous mouse GM-CSF gene with a human GM-CSF gene(second type) is provided, wherein the genetically modified mouse whenengrafted with human hematopoietic cells is capable of infection with S.typhi.

The data shown in FIG. 1 is representative of both the first and thesecond type of mouse. Genetic modifications of the mice in FIG. 1comprise: (a) a mouse RAG gene knockout; and (b) a mouse II2rg geneknockout. The FIG. 1 mice also comprise an engraftment of humanhematopoietic cells. The mice may be further modified by two furthermodifications to create the second type of mouse, which are: (c)replacement of an endogenous mouse IL-3 gene with a human IL-3 gene; and(d) replacement of a mouse GM-CSF gene with a human GM-CSF gene.

FIGS. 2, 3 and 4 were obtained using only the first type of modifiedmice (comprising the modifications (a) a mouse RAG gene knockout; and(b) a mouse II2rg gene knockout; and engraftment with humanhematopoietic cells).

In various embodiments, the S. typhi-infected genetically modified mousecomprises a productive infection of S. typhi. In one embodiment, themouse is capable of harboring and reproducing S. typhi in one or more ofits cells. In one embodiment, the mouse is capable of maintaining a S.typhi titer or level in its blood or in at least one tissue for at leasta week, 10 days, two week, three weeks, or four weeks following aninfective exposure to S. typhi.

In one embodiment, the method comprises administering an agent to agenetically modified mouse in accordance with the invention, wherein thegenetically modified mouse is infected with S. typhi; detecting a levelof S. typhi in blood or a tissue of a mouse following administration ofthe agent, and, optionally, determining if administration of the agentdecreases the level of S. typhi in the blood or tissue of the mouse. Inone embodiment, the agent is a vaccine. In another embodiment, the agentis an antibiotic or an agent that is suspected to have antibioticproperties. In one embodiment, the agent is antigen-binding protein, ina specific embodiment an antibody. In one embodiment, the agent is anapproved pharmaceutical for use in a human.

In one embodiment, the method comprises infecting a genetically modifiedand engrafted mouse in accordance with the invention with a known amountof S. typhi, administering an agent to the infected mouse, anddetermining the amount of S. typhi in the genetically modified andengrafted mouse following administration of the agent. In oneembodiment, the agent is determined to be an anti-S. typhi agent if itreduces the amount of S. typhi in blood or a tissue of the mouse by atleast half following a single administration or two or moreadministrations of the agent over a selected period of time.

In one aspect, a method is provided for determining if a S. typhiisolate or strain of interest is drug resistant or multi-drug resistant,comprising administering a drug or a combination of drugs employed totreat S. typhi to a genetically modified and engrafted mouse accordingto the invention, wherein the mouse is infected with the S. typhiisolate or strain of interest. The method includes determining theeffect, if any, of the drug or combination of drugs on (a) the titer ofthe S. typhi isolate or strain of interest in the blood or tissue of themouse at a point in time after administration of the drug or combinationof drugs, (b) the ability of the S. typhi isolate or strain of interestto maintain an infection in the mouse or a level of S. typhi in a tissueof the mouse after one or more administration(s) of the drug orcombination of drugs, or (c) the ability of the S. typhi isolate orstrain of interest to reproduce in the mouse at a point in time afteradministration of the drug or combination of drugs. In a specificembodiment, the drug is selected from the group consisting ofamoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime,chloramphenicol, ciprofloxacin, co-trimoxazole, ertapenem, imipenem,fluoroquinolones (e.g., ciprofloxacin, gatifloxacin, ofloxacin),streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, and acombination thereof. In a specific embodiment, the administration of thedrug or combination of drugs is at least a week, 10 days, two week,three weeks, or four weeks after an infection-producing exposure to S.typhi.

In various aspects and embodiments, level of S. typhi in blood or tissueis measured by ascertaining the number of colony forming units per unit(e.g., weight or volume) of blood or tissue.

In one embodiment, a genetically modified and human hematopoieticcell-engrafted mouse in accordance with the invention has a S. typhilevel, as measured by colony forming units (cfu's), of at least 100-,1,000-, or 10,000-fold over a mouse that is not engrafted with humanhematopoietic cells.

Methods and compositions useful for ascertaining the efficacy of ananti-S. typhi vaccine. In one aspect, a method for ascertaining theefficacy of an anti-S. typhi vaccine is provided, comprising exposing agenetically modified and engrafted mouse in accordance with theinvention to an anti-S. typhi vaccine, and thereafter exposing thegenetically modified and engrafted mouse to S. typhi, and determiningwhether or to what extent the genetically modified and engrafted mouseis infectable by S. typhi.

In one embodiment, the anti-S. typhi vaccine comprises a S. typhi cellsurface protein or immunogenic fragment thereof. In one embodiment, thevaccine comprises a membrane fraction of a S. typhi strain. In oneembodiment, the vaccine comprises a recombinant S. typhi protein orimmunogenic fragment thereof. In one embodiment, the vaccine comprisesan expression vector that encodes a S. typhi protein or immunogenicfragment thereof. In one embodiment, the vaccine comprises aninactivated S. typhi strain or inactivated mixture of S. typhi strains.

Genetically modified and engrafted mice described in this disclosure arealso useful for modeling human pathogen infections more closely thanexisting mice. For example, infection by M. tuberculosis. Geneticallymodified and engrafted mice described herein are useful for modeling ahuman infection of a mycobacterium, for example, by providing a M.tuberculosis mouse model that develops granulomas, including granulomasthat comprise human immune cells and well-defined granulomas. Themethods for drug and vaccine testing mentioned in connection with S.typhi infection of genetically modified and engrafted mice described arealso applicable to M. tuberculosis applications, e.g., identifyingdrug-resistant strains, testing efficacy of an M. tuberculosis vaccine,testing anti-M. tuberculosis agents, measuring cfu's in response to ananti-M. tuberculosis agent, etc.

Genetically modified and engrafted mice described in this disclosure arealso useful for modeling a human hematopoietic malignancy thatoriginates from an early human hematopoietic cell, e.g. from a humanhematopoietic or progenitor cell. Further applications of thegenetically modified and engrafted mice described in this disclosurewill be apparent to those skilled in the art upon reading thisdisclosure.

Thrombopoietin and Engraftment

Thrombopoietin (TPO) was initially identified as a growth factor thatpromotes the development of megakaryocytes and platelets (Wendling, F.et al. (1994) cMpl ligand is a humoral regulator ofmegakaryocytopoiesis, Nature 369:571-574; Kaushansky, K. et al. (1994)Promotion of megakaryocyte progenitor expansion and differentiation bythe c-Mpl ligand thrombopoietin, Nature 369:568-571; Lok, S. et al.(1994) Cloning and expression of murine thrombopoietin cDNA andstimulation of platelet production in vivo, Nature 369:565-568; deSauvage, F. J. et al. (1994) Stimulation of megakaryocytopoiesis andthrombopoiesis by the c-Mpl ligand, Nature 369:533-538; Bartley, T. D.et al. (1994) Identification and cloning of a megakaryocyte growth anddevelopment factor that is a ligand for the cytokine receptor Mpl, Cell77:1117-1124; Kaushansky, K. (1998) Thrombopoietin, N Engl J Med339:746-754; Kaushansky, K. (2005) The molecular mechanisms that controlthrombopoiesis, J Clin Invest 115:3339-3347; Kaushansky, K. (2008)Historical review: megakaryopoiesis and thrombopoiesis, Blood111:981-986).

TPO is constitutively produced by the liver and the kidneys and releasedinto the blood circulation. The receptor for TPO, c-Mpl, is expressed byhematopoietic stem and progenitor cells in the bone marrow. C-Mpl isalso expressed on circulating platelets. However, the binding of TPO onplatelets does not activate any signaling pathway. Thus, thrombocytesact as a sink or scavengers for TPO and via this mechanism contribute tonegative regulation of thrombopoiesis (Kuter, D. J. & Rosenberg, R. D.(1995) The reciprocal relationship of thrombopoietin (c-Mpl ligand) tochanges in the platelet mass during busulfan-induced thrombocytopenia inthe rabbit, Blood 85:2720-2730). Subsequently, TPO has been recognizedfor its important function to support the expansion and self-renewal ofHSCs (Fox, N., et al. (2002) Thrombopoietin expands hematopoietic stemcells after transplantation, J Clin Invest 110, 389-3894; Kirito, K. etal. (2003) Thrombopoietin stimulates Hoxb4 expression: an explanationfor the favorable effects of TPO on hematopoietic stem cells, Blood102:3172-3178).

TPO deficiency leads to reduced numbers of HSCs in adult mice, and thepresence of TPO is needed to maintain adult HSCs in quiescence(Yoshihara, H. et al. (2007) Thrombopoietin/MPL signaling regulateshematopoietic stem cell quiescence and interaction with the osteoblasticniche, Cell Stem Cell 1, 685-697; Qian, H. et al. (2007) Critical roleof thrombopoietin in maintaining adult quiescent hematopoietic stemcells, Cell Stem Cell 1:671-684). Furthermore, TPO is required tosupport post-transplantation expansion of HSCs, necessary to replenishthe hematopoietic compartment of irradiated hosts. Interestingly, it hasbeen demonstrated that osteoblastic cells involved in forming the HSC“niche” in the bone marrow produce TPO, critical for HSC function andmaintenance.

Although mouse and human TPO are both-sided cross-reactive to therespective cognate receptors when used at supraphysiological doses invitro, affinity and biologic activity might differ when the cytokineacts at limiting, physiological doses in context of an in vivoenvironment. The inventors hypothesized that mouse TPO might not providean appropriate stimulus to the human c-Mpl receptor in vivo, andtherefore could account for the impaired properties of human HSCs in themouse environment. To correct this potential defect, the inventorsreplaced the gene that encodes mouse TPO by its human counterpart inRag2^(−/−)γ_(c) ^(−/−) mice. It was hypothesized that such a mouse wouldhave an improved ability to sustain differentiation and function of ahuman hemat-lymphopoietic system.

Significant progress has been achieved in the development of mice thatsustain differentiation and function of the human hemato-lymphopoieticsystem since the publication of the first models more than two decadesago. However, several limitations remain, including (i) the transienthuman cell engraftment, not lasting for the life of recipient mice, (ii)the unphysiologic bias towards lymphoid lineage and poor differentiationof myeloid cells, and (iii) the variability of engraftment levelsbetween different animals, even when groups of mice are transplantedwith cells from a single human donor. These limitations might be due tonon-physiologic location of human cells, residual xenoreactivity of theimmunodeficient host, different composition of hemato-lymphoid cells inmouse and human species, and/or due to lack or insufficient mouse tohuman cross-reactivity of hematopoiesis supporting factors, leading topreferential mouse cell support. Thus, providing physiologic levels ofhuman growth factors and deleting respective mouse homologues in thehost might further favor the development and survival of human cellpopulations. Here, we describe a novel strain of recipient mice in whichwe humanized the gene encoding thrombopoietin, a cytokine with importantfunctions in the maintenance and self-renewal of hematopoietic stemcells.

Upon engraftment of these humanized thrombopoietin mice with human CD34+hematopoietic stem and progenitor cells, a significant improvement wasobserved compared to previously available models on all threelimitations listed above: bone marrow chimerism was higher and wasmaintained for at least six months; multilineage, in particular myeloidlineage differentiation was enhanced; and variability in engraftmentlevels was reduced.

A major difference between the mouse and human immune systems is thefraction of granulocytes present in the blood. Lymphocytes arepreponderant in mice, while human blood is rich in granulocytes, aspecies difference unclear in its significance. Interestingly, thepresence of human TPO improved differentiation of human granulocytes(FIG. 14). Thus, the presence of human TPO in recipient mice favors abalance between granulocytes and lymphocytes that reflects better thehuman physiological condition, a finding possibly due to bettermaintenance and/or differentiation of human myeloid progenitor cells.

More importantly, the results show that TPO humanization favors themaintenance of secondary recipient repopulating human hematopoietic stemand progenitor cells in the mouse environment (FIG. 15). Hence, theRag2^(−/−)γc^(−/−)TPO^(h/h) mouse represents a novel model to studyvarious aspects of human stem and progenitor cell function in vivo.

Nevertheless, although a better balance between the myeloid and lymphoidlineages in the blood was observed, no significant effect of TPOhumanization on the overall engraftment levels in peripheral lymphoidtissues (including spleen, blood and thymus) was observed (FIG.13(g)-(i)). This could be explained by different factors. First,although the recipient mice are sub-lethally irradiated beforetransplantation, a large population of mouse myeloid cells is stillpresent. Among those cells, macrophages are able to phagocyte humancells and limit the overall engraftment levels in the periphery. Thus,the genetic depletion of mouse macrophages, or their functionalinactivation, might permit higher levels of peripheral engraftment.Second, human cells may require additional human growth factors to favortheir terminal differentiation, egress from the bone marrow and/or theirsurvival in the periphery. A diverse panel of cytokines can beconsidered for each lineage. Finally, although secondary lymphoid organsare formed in humanized mice, their structure is not optimal compared tohuman tissues. This partially defective structure might represent alimit to the number of human cells that can survive in these organs.

Additional gene replacements can be used to further improve the mouserecipients. To achieve this, the technology used in this study, based onthe knock-in replacement of a mouse gene by its human homolog, presentstwo main advantages compared to classical transgenic approaches. First,as it maintains most of the regulatory sequences of mouse origin, itensures that the humanized gene is faithfully expressed in the mousehost. Second, as the knock-in strategy replaces the mouse cytokine byits human homolog, it can affect the population(s) of cells of mouseorigin that depend on this cytokine, in the case that the human cytokineis not fully cross-reactive on the mouse receptor. This can provide afurther competitive advantage to the human cell population(s) aftertransplantation. Indeed, this seems to be the case for human TPO, as thehomozygous replacement of TPO leads to a reduction in mouse plateletsand HSCs in non-engrafted animals (FIGS. 13(a), 15(a) and 15(b)).

With human TPO knock-in mice, an improved model is provided that can beuseful to study in vivo physiology of human hematopoiesis in general andhuman hematopoietic stem and progenitor cells in particular. Moreover,these mice sustain in vivo human hematopoietic malignancies thatoriginate from early hematopoietic cells, such as, e.g., myeloidleukemias and myeloproliferative neoplasias.

EXAMPLES

The following examples are not intended to limit the scope of what theinventors regard as their invention. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in the Celsius scale, and pressure is at or nearatmospheric.

Example 1 Making Human IL-3/GM-CSF and Human TPO Mice

hIL-3/GM-CSF Targeting.

A targeting construct for replacing a mouse IL-3 gene with a human IL-3gene and a mouse GM-CSF gene with a human GM-CSF gene in a singletargeting step was constructed using VELOCIGENE® technology (see, e.g.,U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) “High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis,” Nat Biol 21(6):652-659; hereby incorporated by reference)employing gap repair cloning.

Mouse sequences were obtained from bacterial artificial chromosome (BAC)RPCI-23, clone 5E15. The human sequences were obtained from Caltech Dlibrary (CTD), BAC clone 2333J5.

A gap repair donor vector containing a p15 origin of replication wasconstructed by cloning a 5′ mouse homology arm immediately upstream ofthe mIL-3 ATG, a human 5′ IL-3 homology arm extending from the hIL-3 ATGto about 274 nts into the hIL-3 gene, a poly linker, a 3′ hGM-CSFbeginning about 2.9 kb downstream of the polyA sequence of the hGM-CSFgene (about 233 bases), and a loxed drug selection cassette followed bya mouse 3′ homology arm having sequence downstream (about 2.9 kbdownstream) of the mGM-CSF polyA sequence. The gap repair vector waslinearized and inserted into E. coli strain DH10B containing the humanCTD BAC clone 2333J5 and a recombination enzyme vector as described inValenzuela et al.

Cells were grown in drug selection medium. Individual clones were grown,gap repair donor vector DNA was extracted, and portions of the vectorwere sequenced for proper mouse-human junctions. Pulsed field gelelectrophoresis was used to establish insert size and expectedrestriction fragment length.

Captured donor containing mouse upstream and downstream homology boxesflanking the hIL-3 gene, the hGM-CSF gene, and the loxed drug selectioncassette was obtained from repair donor vector, the captured donor waslinearized, and linearized captured donor was introduced into E. coliDH10B containing RPCI23 clone 5E15 and pABG vector. Cells were grown indrug selection medium. Individual clones containing captured donor DNAin RPCI23 clone 5E15 DNA (to form the targeting vector) were isolated,targeting vector DNA was extracted, and portions of the vector weresequenced for proper mouse-human junctions. Pulsed field gelelectrophoresis was used to establish insert size and expectedrestriction fragment length.

Electroporation.

The targeting vector was linearized and used to electroporate mouse EScells as described in Valenzuela et al. Electroporated mouse ES cellscontaining the targeting vector were further electroporated with atransient Cre-expressing vector to remove the loxed drug selectioncassette. The targeting vector was electroporated into Rag2 HET II2rgy/− ES cells. The parental ES cell line in which the RAG2 gene and II2rggene knockout was made was a commercially available V17 ES cell(BALB/c×129 heterozygote). ES cells targeted with the hIL-3 and hGM-CSFgenes were used to introduce into mouse embryos.

hIL-3/GM-CSF Mice.

Targeted donor ES cells are introduced into an 8-cell stage mouse embryoby the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 andPoueymirou et al. (2007) “F0 generation mice that are essentially fullyderived from the donor gene-targeted ES cells allowing immediatephenotypic analyses,” Nat Blot 25(1):91-99; hereby incorporated byreference). VELOCIMICE® (F0 mice fully derived from the donor ES cell)bearing the humanized IL-3 and GM-CSF, constructs are identified bygenotyping for loss of mouse allele and gain of human allele using amodification of allele assay (see, e.g., Valenzuela et al.). These miceare first bred with BALB/cAnNCR and then mice heterozygous for Rag2 andII2rg plus the human IL-3/GM-CSF KI are bred with Rag2/II2rg double KOmice for engraftment studies.

Phenotyping hIL-3/GM-CSF Mice.

Humanized mice were tested for production of human GM-CSF by RT-PCRusing hGM-CSF-specific primers. The expression pattern of human GM-CSFfor the tissues tested matched that of mouse GM-CSF (primarilyexpression in lung). ELISAs of splenocytes stimulated with ConA and IL-2for 48 hours from the humanized mice were done to detect the presence ofhIL-3 and hGM-CSF; splenocytes were positive for expression of bothhIL-3 and hGM-CSF.

hTPO Targeting.

A targeting construct (FIG. 11(d)) for replacing the mouse Tpo (mTpo)with the human TPO (hTPO) gene in a single targeting step wasconstructed using VELOCIGENE® technology employing gap repair cloning(Valenzuela et al.). The vector was designed to replace the sequenceencompassing the open reading frame of Tpo, but to maintain the promoterand 5′UTR of mouse origin. Mouse sequences were obtained from bacterialartificial chromosome (BAC) RPCI-23, clone 98H7. Human sequences wereobtained from BAC RPCI-11, clone 63 m3. A gap repair donor vectorcontaining a p15 origin of replication was constructed by cloning a 5′mouse homology arm immediately upstream of the mTpo ATG, a human 5′ TPOhomology arm extending from the hTPO ATG to about 275 nts into the hTPOgene, a poly linker, a 3′ hTPO homology arm beginning about 1.5 kbdownstream of the polyA sequence of the hTPO gene, and a loxed drugselection cassette followed by a mouse 3′ homology arm having sequencedownstream (about 3.5 kb downstream) of the mTpo polyA sequence. The gaprepair vector was linearized and inserted into E. coli strain DH10Bcontaining the human BAC clone RPCI-11, 63 m3 and a recombination enzymevector. Cells were grown in drug selection medium. Individual cloneswere grown, gap repair donor vector DNA was extracted, and portions ofthe vector were sequenced for proper mouse-human junctions. Pulsed fieldgel electrophoresis was used to establish insert size and expectedrestriction fragment length. Captured donor containing mouse upstreamand downstream homology boxes flanking the hTPO gene and the loxed drugselection cassette was obtained from repair donor vector, the captureddonor was linearized, and linearized captured donor was introduced intoE. coli DH10B containing RPCI-23 clone 98H7 and pABG vector. Cells weregrown in drug selection medium. Individual clones containing captureddonor DNA in RPCI-23 clone 98H7 DNA (to form the targeting vector) wereisolated, targeting vector DNA was extracted, and portions of the vectorwere sequenced for proper mouse-human junctions. Pulsed field gelelectrophoresis was used to establish insert size and expectedrestriction fragment length. The targeting vector was linearized andused to electroporate mouse embryonic stem (ES) cells. The targetingvector was electroporated into RAG2+/− γcY/− ES cells. The parentalRAG2^(+/−) γc^(Y/−) ES cell line was made from a commercially availableV17 ES cell line (BALB/c×129 heterozygote). Correctly targeted ES cellswere further electroporated with a transient Cre-expressing vector toremove the loxed drug selection cassette. ES cells targeted with thehTPO gene and without selection cassette were introduced into an 8-cellstage mouse embryo by the VELOCIMOUSE® method (Poueymirou et al.).Rag2^(−/−)γ_(c) ^(−/−) mice with wild-type Tpo (TPO^(m/m)), heterozygous(TPO^(h/m)) or homozygous (TPO^(h/h)) TPO gene replacement wereobtained.

Example 2 hIL-3/GM-CSF Mice Engraftment

Isolation of Human Hematopoietic Stem Cells.

Human umbilical cord blood and fetal liver samples were obtained underapproval from the Yale University Human Investigation Committee fromYale-New Haven Hospital and Albert Einstein Medical College New York,respectively. CD34+ cells were isolated from human umbilical cord bloodor fetal liver by density gradient centrifugation and immunomagneticselection using CD34 microbeads (Miltenyi Biotec). Purity of isolatedCD34-positive cells was verified by flow cytometry. Purified humanCD34-positive cells were cryopreserved and stored in liquid nitrogenbefore use.

Engraftment of Mice with Human Hematopoietic Stem Cells.

Engraftment was done as previously described (Traggiai et al. (2004)Development of a human adaptive immune system in cord bloodcell-transplanted mice, Science 304:104-107). Briefly, on the day ofbirth pups from RAG2 gene knockout/II2rg gene knockout background (withor without hIL-3/hGM-CSF) were sublethally irradiated (2×200 cGy with a4 hour interval). After irradiation newborn pups received 1-2×10⁵ humanCD34+ cells (resuspended in 25 microliters of PBS) by intrahepaticinjection using a 30-gauge needle. Controls were injected with PBS only.Mice were weaned at 3-4 weeks of age and maintained under specificpathogen-free conditions. Mice received prophylactic antibiotics(Sulfatrim) in the drinking water to prevent opportunistic infections.All animal work was approved by Yale University Institutional AnimalCare and Use Committee (IACUC) and conducted in accordance with IACUCregulations.

Analysis of Engrafted Mice.

Engraftment with human hematopoietic cells was determined 8-12 weekspost-transplantation. Blood samples were obtained from the retro-orbitalsinus and lysis of red blood cells was performed using ACK lysis buffer(Lonza). Samples were then stained with fluorescently labeled monoclonalantibodies against mouse CD45, human CD45, human CD3, and human CD14(all from BD Biosciences) and analyzed by flow cytometry on aFACScalibur™ (BD Biosciences). Mice used for infection experiments hadblood engraftment levels of >4% hCD45+ cells unless indicated otherwise.Matched mice, i.e., mice engrafted with the same batch of CD34+ cells,were used for experiments. Unless indicated otherwise, experiments wereperformed with mice engrafted with CD34+ cells from FL.

Flow Cytometry.

For hIL-3/GM-CSF studies, cell suspensions were prepared from lung, BAL,bone marrow, thymus, spleen and blood of mice 10-14 weekspost-transplantation. Lysis of RBC was performed using ACK lysis buffer(Lonza). Samples were then stained with fluorochrome-labeled monoclonalantibodies (mAbs) against mouse and human cell surface antigens. Thefollowing mAbs were used: (1) Anti-human: CD3 (UCHT1), CD4 (RPA-T4), CD8(HIT8a), CD11c (B-ly6), CD14 (MoP9), CD19 (HIB19), CD33 (WM53), CD45(HI30 and 2D1), CD56 (NCAM 16.2), CD66 (B1.1), CD116 (4H1), CD123 (9F5).(2) Anti-mouse: CD45 (30-F11), F4/80 (BM8). CD116, CD45 (30-F11), andF4/80 mAbs were from eBioscience. All other mAbs were from BDBiosciences. Samples were analyzed on a FACSCalibur™ or LSRII™ flowcytometer (BD Biosciences).

Methylcellulose CFU Assay.

For hIL-3/GM-CSF studies, human CD34+ bone marrow cells from engraftedmice were purified by cell sorting. Sorted cells (1-1.5×10⁵) werecultured in Iscove's modified Dulbecco's medium (IMDM, GIBCO) basedmethylcellulose medium (Methocult™ H4100, StemCell Technologies) thatwas supplemented with 20% FBS, 1% BSA, 2 mM L-glutamine, 55 μM2-mercaptoethanol and the following human cytokines: stem cell factor(10 ng/ml), FLT3 ligand (10 ng/ml), thrombopoietin (50 ng/ml), IL-3 (20ng/ml), IL-6 (10 ng/ml), IL-11 (10 ng/ml), GM-CSF (50 ng/ml), anderythropoietin (4 U/ml) (all R & D Systems). Cells were incubated in 60mm Petri dishes at 37° C./5% CO₂. The number of colonies was determinedby microscopy after 12-14 days.

Inflammatory Response to LPS.

Mice received two i.p. injections of Ultrapure LPS E. coli 0111:B4(Invivogen) 48 h apart (35 and 17.5 μg). Sera were harvested 2-3 h aftereach injection. Serum concentrations of human IL-6 were determined byELISA (R&D Systems). Mice were sacrificed 72 h after the first LPSinjection, blood collected by cardiac puncture and used for flowcytometry.

Intracellular Cytokine Staining.

For hIL-3/GM-CSF studies, overlapping peptides covering the whole TB10.4protein (Skjot et al. (2002) Epitope mapping of the immunodominantantigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, whichconstitute a subfamily of the esat-6 gene family, Infect. Immun.70:5446-5453) were synthesized by the W.M. Keck Facility of YaleUniversity. Splenocytes from BCG-infected mice (2×10⁶/well) wereincubated with mixed peptides (each peptide at 5 μg/ml) in a totalvolume of 200 μl/well in 96-well U-bottom microtiter plates (BectonDickinson) for 5 h at 37° C./5% CO₂. RPMI 1640 medium (Invitrogen)supplemented with 10% FCS, 1% penicillin-streptomycin, 1% L-glutamine,and 55 μM 2-mercaptoethanol was used for cell culture. Intracellularcytokine staining was performed using the Cytofix/Cytoperm™ kit (BDBiosciences) according to the manufacturer's instructions. The followingmAbs were used for intracellular staining (all BD Biosciences):Anti-human IFNγ (B27), anti-mouse IFNγ (XMG1.2). Isotype-matched mAbswere used as controls.

Histology and Immunohistochemistry.

For hIL-3/GM-CSF studies, organs were harvested and fixed in 10%neutral-buffered formalin or Zinc Fixative (BD Biosciences) forhistological analysis. Paraffin-embedded tissues sections were prepared,stained with H&E or PAS, or processed for immunohistochemistry by theYale Pathology Tissue Services. The following anti-human Abs were usedfor immunohistochemistry (all from Dako): CD45 (2B11+PD7/26), CD3(F7.2.38), CD68 (PG-M1). Scoring of tissue sections for the presence ofgranulomas was performed in a blinded fashion.

Statistical Analysis.

For hIL-3/GM-CSF studies, the non-parametric Mann-Whitney U test wasused to determine statistical significance between two groups (α=0.05).For multigroup comparisons, we applied one-way ANOVA with post hoctesting using Tukey's Multiple Comparison Test (α=0.05). Onlystatistically significant P values (P<0.05) are shown.

Example 3 hIL-3/GM-CSF Engrafted Mice Infection

S. typhi ISP2825 (Galán J. E. & Curtiss, R. (1991) Distribution of theinvA, -B, -C, and -D genes of S. thyphimurium among other S. serovars:invA mutants of S. typhi are deficient for entry into mammalian cells,Infect. Immun. 59(9):2901-2908, hereby incorporated by reference), aclinical isolate from a patient suffering from typhoid fever, was grownovernight in LB broth. On the following day, 40 microliters of thebacterial cell culture was transferred into 2 mL of fresh LB brothcontaining 0.3M NaCl and grown for ˜3 hrs at 37° C. until the culturereached an OD₆₀₀ of ˜0.9. The bacterial culture was spun down,resuspended in buffered saline solution, and used for infections. Nineto twelve week old humanized mice and control mice were inoculated onday 0 intraperitoneally with 1×10³ or 1×10⁴ or 1×10⁵ of S. typhi. Theinfected mice were closely monitored and sacrificed at 4 weeks postinfection. Spleen, liver, and gallbladder were aseptically removed andmechanically homogenized in 3-5 mL of sterile PBS containing 0.05%sodium deoxycholate. The tissue homogenate was serially diluted, platedon LB agar plates, and incubated overnight at 37° C. for colony counts.Colonies were counted and the number of total colony-forming unitsrecovered was calculated. Mouse data are provided in FIGS. 1-4. In FIG.1, “Control” mice are unengrafted genetically modified mice (RAG KO,Il2rg KO/hIL-3, hGM-CSF). In FIGS. 2-4, “Control” mice are unengraftedmice with a RAG KO and an Il2rg KO (i.e., they lack humanization of IL-3and GM-CSF, but instead have endogenous mouse IL-3 and endogenous mouseGM-CSF). “Control” mice were injected with PBS instead of human CD34+cells.

As shown in FIG. 1, S. typhi infection in spleen is detected in the twogenetically modified mice (RAG KO, Il2rg KO/hIL-3, hGM-CSF) at 10 dayspost-infection.

As shown in FIG. 2, at one week post-infection with 1×10³ S. typhi,genetically modified mice (RAG KO, Il2rg KO) with percent engraftmentsof 3.8 and 3 showed infection in spleen (p<0.01), at about athousand-fold higher than control mice. The p value for differencebetween Control and Humanized was p<0.01.

As shown in FIG. 3, genetically modified mice (RAG KO, Il2rg KO)engrafted with CD34-positive cells from fetal liver and infected with1×10⁴ S. typhi at four weeks post-infection showed S. typhi infection inboth spleen (an average of about 1,000- to about 10,000-fold) and liver(an average of about 1,000-fold). Individual mice in the cohort testedfor spleen S. typhi had (from top to bottom in the “Humanized” cohort inthe left panel of FIG. 3) percent engraftment of human cells of 23.5,40.1, 16.5, 50, 26, and 51.7. Individual mice in the cohort tested forliver S. typhi had (from top to bottom in the “Humanized” cohort in theright panel of FIG. 3) percent engraftment of human cells of 16.5, 40.1,23.5, 26, 50, and 51.7. Thep value for difference between Control andHumanized in spleen was p<0.01; in liver p<0.03.

As shown in FIG. 4, genetically modified mice (RAG KO, Il2rg KO)engrafted with CD34-positive cells from fetal liver and infected with1×10⁴ S. typhi at four weeks post-infection showed S. typhi infection ingall bladder, with a S. typhi cfu of, on average, a million-fold higherthan the control mouse. Individual mice in the cohort tested for gallbladder S. typhi had (from top to bottom in the “Humanized” cohort inFIG. 3) percent engraftment of human cells of 23.5, 16.5, 40.1, 50, 26,and 51.7. Thep value for difference between Control and Humanized wasp<0.03.

The results establish that the genetically modified mice (RAG KO, II2rgKO/hIL-3, hGM-CSF) can be colonized by S. typhi after systemicinfection.

Example 4 hIL-3/GM-CSF Engrafted Mice Validation of Mouse Model forHuman Inflammatory Responses to Lung Pathogens

The genes encoding GM-CSF (Csf2) and IL-3 are closely linked (<10 kb) onchromosomes 5 and 11 in humans and mice, respectively. This allowedreplacement of the mouse with the human loci for both genes to generatehIL-3/GM-CSF KI mice (FIG. 5(e)). While the human II3 KI allele is underthe control of mouse regulatory elements, the human Csf2 KI alleleremains under the control of its human regulatory elements. Expressionof mouse and human GM-CSF mRNA was analyzed by RT-PCR in hIL-3/GM-CSF KImice expressing one allele of each mouse and one allele of each humangene, referred to as IL-3/GM-CSF “human/mouse” (h/m) mice. Wild-typemice that only have the mouse alleles of IL-3 and GM-CSF are referred toas IL-3/GM-CSF “mouse/mouse” (m/m) mice.

RT-PCR and ELISA Analysis of hIL-3/GM-CSF KI Mice.

Total RNA was extracted from homogenized tissues with TRIzol™ reagent(Invitrogen) according to the manufacturer's instructions. Equal amountsof DNase-treated RNA were used for cDNA synthesis with the SuperScript™First-Strand Synthesis System (Invitrogen). Conventional RT-PCR wasperformed with the following primers: (1) Mouse Csf2: forward,CCAGTCCAAA AATGAGGAAG C (SEQ ID NO:7); reverse, CAGCGTTTTC AGAGGGCTAT(SEQ ID NO:8). (2) Human Csf2: forward, GGCGTCTCCT GAACCTGAGT (SEQ IDNO:9); reverse, GGGGATGACA AGCAGAAAGT (SEQ ID NO:10). (3) Mouse RpI13:forward, GTACGCTGTG AAGGCATCAA (SEQ ID NO:11); reverse, ATCCCATCCAACACCTTGAG (SEQ ID NO:12). Quantitative RT-PCR was performed on a 7500Fast Real-Time PCR system with primer-probe sets purchased from ABI.Expression values were calculated using the comparative threshold cyclemethod and normalized to mouse or human HPRT. Mouse and human IL-3 andGM-CSF protein were detected with species-specific ELISA kits from R&DSystems according to the manufacturer's instructions. Splenocytes wereactivated with 5 μg/ml Concanavalin A (ConA) and 100 U/ml IL-2 andsupernatants harvested for ELISA after 48 h of stimulation.

Expression in hIL-3/GM-CSF KI Mice.

Human GM-CSF mRNA was expressed in a similar pattern to its mousecounterpart with highest expression in the lung (FIG. 5(a)). IL-3 isexpressed mainly by activated T cells that also produce GM-CSF.Therefore, ELISA was performed on supernatants from activatedsplenocytes isolated from h/m mice; both human IL-3 and GM-CSF proteincould be detected (FIG. 6(f) f, 6(g)). To confer a competitive advantageto human hematopoietic cells, generated homozygous KI mice weregenerated that express two alleles of human IL-3 and GM-CSF, referred toas IL-3/GM-CSF “human/human” (h/h) mice. Conventional and quantitativeRT-PCR analysis of lung tissue showed that h/h mice express onlyhuman—but not mouse—GM-CSF mRNA (FIG. 5(b), 5(c)). Human GM-CSF proteincould be detected by ELISA in the bronchioalveolar lavage (BAL) fluid ofh/h mice (FIG. 5(d)). The results show that hIL-3/GM-CSF KI micefaithfully express human GM-CSF (and IL-3).

FIG. 5(a)-(d) shows validation of human GM-CSF expression innon-engrafted hIL-3/GM-CSF KI mice. FIG. 5(a) shows representativeRT-PCR analysis of GM-CSF mRNA expression in various tissues from KImice with one allele of human and one allele of mouse Csf2 (h/m). Li,liver; Br, brain; Lu, lung; Mu, muscle; Sp, spleen; Th, thymus; LN,lymph node; BM, bone marrow. Bottom, specificity of primers to detecthuman GM-CSF was verified by RT-PCR analysis of tissues from controlmice (m/m). Ribosomal protein L13 (RpI13) served as an endogenouscontrol. FIG. 5(b) shows RT-PCR analysis of GM-CSF mRNA expression inlungs from m/m mice or homozygous KI mice expressing two alleles ofhuman Csf2 (h/h) (each n=5). RpI13 served as an endogenous control; NTC,no template control. FIG. 5(c) shows quantitative RT-PCR analysis ofGM-CSF mRNA expression as in (b); GM-CSF expression was normalized tomouse Hprt (each n=5). FIG. 5(d) shows ELISA of human GM-CSF protein inBAL fluid recovered from m/m or h/h KI mice (each n=6); results arerepresentative of two independent experiments; each dot represents onemouse; horizontal bars indicate mean values.

FIG. 5(e) shows a strategy to generate hIL-3/GM-CSF KI mice; genomicorganization of mouse (top) and human (bottom) II3 and Csf2 loci areshown on chromosomes 11 and 5, respectively. Mouse loci were replacedwith human loci as described in this disclosure.

FIG. 6(f),(g) shows expression of human IL-3 and GM-CSF in non-engraftedhIL-3/GM-CSF KI mice. ELISA results for mouse and human IL-3 (f) andGM-CSF (g) production by activated splenocytes are presented.Splenocytes from either m/m (open bars) or h/m KI mice (filled bars)were stimulated with ConA and IL-2 for 48 h and supernatants harvested(each n=1). Human IL-3 and GM-CSF were not detectable (ND) in m/m mice.

Example 5 hIL-3/GM-CSF Engrafted Mice Enhanced Human InflammatoryResponses

hIL-3/GM-CSF KI mice were generated from embryonic stem (ES) cells withone allele of both Rag2 and II2rg already deleted. Breeding onto theRag2 KO II2rg KO background then allowed engraftment with human CD34+hematopoietic cells. Overall human CD45+ hematopoietic cell chimerismand distribution of T, B, and natural killer (NK) cells in bone marrow,thymus, spleen, and blood was not significantly increased inhIL-3/GM-CSF KI mice (data not shown). Also, the frequencies of totalhuman CD33+ myeloid cells, CD66+ granulocytes, CD14+monocytes/macrophages, CD14loCD16+ non-classical monocytes, CD11c+dendritic cells (DC), and CD123+CD11c− plasmacytoid DC were notsignificantly increased in hIL-3/GM-CSF KI mice (data not shown). Thisapplied to both h/m and h/h mice under steady-state conditions. Finally,human bone marrow cells from engrafted hIL-3/GM-CSF KI mice had asimilar capacity to form myeloid colonies in methylcellulose in vitro(data not shown). These findings are consistent with results from KOmouse studies showing that both IL-3 and GM-CSF are largely dispensablefor steady-state myelopoiesis in the organs analyzed here.

In contrast, GM-CSF plays an important role in mediating inflammatoryresponses. GM-CSF expression is induced by inflammatory stimuli, whichleads to the production of inflammatory cytokines (such as IL-6 andTNFα) by monocytes/macrophages and to their recruitment to sites ofinflammation. Human CD14+ monocytes from engrafted hIL-3/GM-CSF KI micehad the highest expression of the GM-CSF receptor α-chain (CD116) (FIG.8(d)). Therefore, the analysis of engrafted hIL-3/GM-CSF KI mice focusedon human monocytes/macrophages. First, the inflammatory response ofhuman monocytes in engrafted hIL-3/GM-CSF KI mice was analyzed. Systemicinflammation was induced by intraperitoneal (i.p.) injection oflipopolysaccharide (LPS). The frequency of circulating human CD14+monocytes was significantly increased in h/m compared to control m/mmice after LPS injection (FIG. 8(e),(f)). Enhanced mobilization of humanmonocytes in h/m mice was associated with increased serum concentrationsof human IL-6 after one and two injections of LPS (FIG. 8(g)).LPS-induced production of human TNFα was also increased in h/m mice, butthis result did not reach statistical significance. These data indicatethat hIL-3/GM-CSF KI mice engrafted with human hematopoietic cells haveenhanced human inflammatory responses mediated by human myelo-monocyticcells.

FIG. 8(d)-(g) illustrates enhanced human inflammatory responses inengrafted hIL-3/GM-CSF KI mice. FIG. 8(d) shows flow cytometry analysisof human bone marrow cells from engrafted hIL-3/GM-CSF h/m KI mice insteady state; the dot plot (left) is gated on hCD45+mCD45− cells. Thehistogram (right) shows GM-CSF receptor α (CD116) expression on CD14−cells (population 1), CD14mid/SSChi granulocytes (population 2), andCD14hi monocytes (population 3). One representative example from a totalof 12 mice analyzed is shown. FIG. 8(e) contains a representative flowcytometry analysis of human blood cells from CB-engrafted m/m or h/m KImice 72 h after two i.p. injections of LPS. Plots are gated onhCD45+mCD45− cells. Numbers next to boxed areas indicate the percentagesof human CD14+ cells. FIG. 8(f) illustrates the frequency of human CD14+blood cells in engrafted m/m (n=4) or h/m KI mice (n=8) 72 h post-LPSinjections. FIG. 8(g) shows ELISA results for human IL-6 in sera fromengrafted m/m (n=4-5) or h/m KI mice (n=8) 2-3 h after first (top) andsecond (bottom) LPS injection. One m/m mouse died after the first LPSinjection. Each dot represents one mouse. Horizontal bars indicate meanvalues. Results are representative of two independent experiments.

Example 6 hIL-3/GM-CSF Engrafted Mice Enhanced Human MacrophageEngraftment in Lung

BAL Analysis.

Brochoalveolar analyses for hIL-3/GM-CSF studies were conducted in thefollowing manner. Lungs were inflated with 1 ml PBS via a catheterinserted into the trachea. This was repeated twice and the recoveredlavage pooled. After centrifugation, cell-free supernatants were savedfor determination of GM-CSF protein concentration by ELISA or for totalprotein content with the BCA Protein Assay Kit (Pierce) according to themanufacturer's instructions. After red blood cell (RBC) lysis with ACKlysis buffer (Lonza), cell pellets were counted and either used for flowcytometry or for cytospin preparations. Cells were spun onto slides andstained with Diff-Quik™ Stain Set (Dade Behring) according to themanufacturer's instructions.

Enhanced Macrophage Engraftment.

The absence of mouse GM-CSF leads to impairment of mouse alveolarmacrophages (AM), which should favor reconstitution with humanmacrophages in homozygous hIL-3/GM-CSF KI mice. In support of this,human GM-CSF is highly expressed in the lung and BAL of h/h mice, whilemouse GM-CSF is lacking.

Mouse AM from non-engrafted h/h mice were enlarged and had the typical“foamy” appearance (FIG. 9(f)) which has been described for AM fromGM-CSF KO mice. GM-CSF KO mice develop PAP due to a defect in surfactantclearance by AM that have a block in terminal differentiation. Similarlyto what has been reported for GM-CSF KO mice, non-engrafted h/h micedeveloped features of PAP such as the subpleural accumulation of AM fullof Periodic acid-Schiff (PAS)-positive material (FIG. 9(g)). It wastherefore concluded that non-engrafted h/h mice show impaireddifferentiation of mouse AM and develop PAP, and are thereforefunctionally equivalent to GM-CSF KO mice.

FIG. 9(f),(g) shows PAP development in non-engrafted homozygoushIL-3/GM-CSF KI mice. FIG. 9(f) shows Diff-Quick™ staining of BAL cellsfrom non-engrafted m/m or h/h KI mice (magnification 400×); onerepresentative example of a total of six mice analyzed per group isshown. FIG. 9(g) shows PAS staining of lung tissue sections fromnon-engrafted m/m or h/h KI mice (magnification 400×); onerepresentative example of a total of 12 mice analyzed per group isshown.

Next, the lung compartment of h/h mice after engraftment with humanhematopoietic cells was examined. FACS analysis showed that h/h mice hadconsiderably more human CD45+ cells in the BAL (FIG. 6(a),(b)).Quantitative RT-PCR of lung tissue revealed that this increase in humancells consisted mainly of cells expressing mRNA for the human myeloidmarkers CD33, CD11b, CD11c, and CD14 (FIG. 6(c)). Furthermore, mRNAexpression of human CD68, a mature macrophage marker that is mainlyexpressed intracellularly, was markedly increased in engrafted h/h mice(FIG. 6(d)). This increase in h/h mice was associated with higherexpression of two transcription factors that are expressed by AM, namelyPU.1 (Spit) and peroxisome proliferator-activated receptor-γ (PPARγ)(FIG. 6(d)). PU.1 is highly expressed in terminally differentiated AM ina GM-CSF-dependent manner. Importantly, transduction of GM-CSF KO AMwith PU.1 in vitro reverses their functional impairment. PPARγ is alsohighly expressed in AM and, similarly to GM-CSF KO mice, PPARγ KO micedevelop PAP. Immunohistological staining of lung sections revealed thepresence of numerous hCD68+ cells with a typical intra-alveolarlocation, consistent with human AM, in engrafted h/h mice (FIG. 6(e)).In contrast, very few human AM could be detected in engrafted m/mcontrol mice. In summary, lungs of CD34+ hematopoietic cell transplantedh/h mice show markedly improved engraftment of human macrophages.

FIG. 6(a)-(e) show that homozygous hIL-3/GM-CSF KI mice have betterhuman macrophage engraftment in the lung. FIG. 6(a) shows representativeflow cytometry analysis of BAL cells from engrafted m/m and h/h KI mice.Numbers next to outlined areas indicate the percentages of hCD45+ andmCD45+ hematopoietic cells. mCD45+hCD45+ cells have highautofluorescence and constitute F4/80+ mouse AM. FIG. 6(b) provides thenumbers of human hematopoietic (hCD45+) cells in BAL from engrafted m/mand h/h KI mice (results are combined from three independent experiment(total n=15 per group)). FIG. 6(c) shows results of quantitative RT-PCRanalysis of human lymphoid and myeloid gene expression in lung tissuefrom engrafted m/m and h/h KI mice (each n=4). Expression was normalizedto mouse HPRT (*, P<0.05). FIG. 6(d) shows quantitative RT-PCR analysisof human macrophage gene expression in lung tissue from engrafted m/mand h/h KI mice (each n=4). Expression was normalized to mouse HPRT (*,P<0.05). Each dot represents one mouse. Horizontal bars indicate meanvalues. FIG. 6(e) shows immunohistochemistry of lung tissue sectionsstained for human CD68 from engrafted m/m and h/h KI mice (magnification100× (top) and 200× (bottom)). One representative example of a total of10 mice analyzed per group is shown.

Example 7 hIL-3/GM-CSF Engrafted Mice PAP Alleviated by HumanHematopoietic Cells

It was investigated whether the increased engraftment of h/h mice withhuman macrophages leads to better human immune function in the lung.First, it was investigated whether human macrophages can rescue the PAPsyndrome that is found in non-engrafted h/h mice. Although both type IIalveolar epithelial cells and AM can respond to GM-CSF, PAP can berescued by bone marrow transplantation. This indicates thathematopoietic cells, specifically AM, are the main cell type being ableto reverse PAP. Therefore, it was hypothesized that h/h mice engraftedwith human hematopoietic cells should have less severe PAP. As expected,non-engrafted h/h mice showed intra-alveolar accumulation ofPAS-positive material (FIG. 7(a)), which is a hallmark of PAP.Consistent with the hypothesis, engrafted h/h mice had less severeprotein accumulation in the lung, with the lungs of some h/h miceresembling (non-engrafted or engrafted) m/m control mice (FIG. 7(a)). Inaddition, engrafted h/h mice had significantly lower amounts of totalprotein in the BAL fluid than non-engrafted h/h mice (FIG. 7(b)). Theseresults indicate that engrafted human hematopoietic cells (presumablyAM) are capable of alleviating PAP in homozygous hIL-3/GM-CSF KI mice.

FIG. 7(a)-(e) show that human hematopoietic cells alleviate PAP inhomozygous hIL-3/GM-CSF KI mice. FIG. 7(a) shows PAS staining of lungtissue sections from non-engrafted or engrafted m/m or h/h KI mice. Lungsections from two different engrafted h/h KI mice are shown(magnification 400×). Representative examples of a total of 10-12 miceanalyzed per group are shown. FIG. 7(b) shows quantification of totalprotein in BAL fluid from non-engrafted (non) or engrafted h/h KI miceor m/m control mice (n=6 per group). P<0.0001 (one-way ANOVA testing).Values of P as determined by Tukey's Multiple Comparison Test areindicated by asterisks (**, P<0.01; ***, P<0.001).

Example 8 hIL-3/GM-CSF Engrafted Mice A stronger Human Type I IFNResponse to Influenza A

Influenza A infection.

Mice (9-10 weeks old) were infected with 2×10⁴ plaque-forming units ofinfluenza A/PR8 (H1N1) virus via the intranasal route. Infection wasperformed by the intranasal application of 50 μl virus stock diluted inPBS (or an equal volume of PBS as a control) to mice that had beendeeply anesthetized with Anafane™ (Ivesco). Lungs were harvested 24 hafter infection for RNA extraction and quantitative RT-PCR analysis asdescribed above.

In addition to their role in lung homeostasis, AM are essential for hostdefense in the lung. Numerous studies have shown that GM-CSF KO mice aremore susceptible to a variety of pathogens in the lung. To assess thefunctional response of engrafted human AM to a lung pathogen, engraftedh/h mice were infected with influenza A/PR8 (H1N1) virus via theintranasal route. AM are the main producers of type I interferons (IFN)after infection with pulmonary viruses and AM are required for aneffective innate response to influenza A. Expression of humanhypoxanthine phosphoribosyltransferase (HPRT) mRNA was significantlyhigher in the lungs of engrafted h/h compared to control m/m mice (FIG.8(a)), indicating better human immune cell chimerism. Engrafted m/m miceshowed no significant induction of human IFNβ mRNA expression afterinfluenza A infection when compared to engrafted m/m mice that hadreceived PBS intranasally (FIG. 8(b)). In contrast, engrafted h/h miceexpressed significantly more human IFNβ mRNA than both non-infected h/hmice and infected m/m mice (FIG. 8(b)). The increased expression ofhuman IFNβ mRNA in h/h mice compared to m/m mice was still significantwhen normalized to human HPRT, i.e., to the number of human cells in thelung (FIG. 8(c)). Taken together, homozygous hIL-3/GM-CSF KI mice allowbetter human macrophage chimerism and function in the lung that leads toenhanced human mucosal immunity to viral infection.

FIG. 8(a)-(g) shows that homozygous hIL-3/GM-CSF KI mice mount astronger human type I IFN response to influenza A infection. FIG.8(a)-(c) show quantitative RT-PCR analysis of gene expression in lungtissue from m/m and h/h KI 24 h after intranasal infection withinfluenza A (PR8) (each n=8). Intranasal application of PBS was used asa control (PBS) (each n=4). FIG. 8(a) shows expression of human Hprtnormalized to mouse Hprt. P<0.0001 (one-way ANOVA testing). FIG. 8(b)shows expression of human IFNγ normalized to mouse Hprt. P=0.0171(one-way ANOVA testing). FIG. 8(c) shows expression of human IFNγnormalized to human Hprt. P=0.0032 (one-way ANOVA testing). Values ofPas determined by Tukey's Multiple Comparison Test are indicated byasterisks (*, P<0.05; **, P<0.01; ***, P<0.001). Each dot represents onemouse. Horizontal bars indicate mean values. Results are representativeof two independent experiments.

Example 9 hIL-3/GM-CSF Engrafted Mice Granulomas with Human Cells afterMycobacterial Infection

The potential of hIL-3/GM-CSF KI mice to support human inflammatoryresponses to a second pathogen with tropism for the lung, wheremacrophages play a central role in the pathogen-specific immuneresponse, was investigated. Granuloma formation after infection withmycobacteria was selected. The granuloma represents a specialized localinflammatory response that is the hallmark of infection withmycobacteria. It is a classic example of a DTH response and itsformation is dependent on the interaction between activated T cells andmacrophages. Both IL-3 and GM-CSF are required for optimal DTH responsesand, importantly, GM-CSF KO mice do not form granulomas when infectedwith mycobacteria.

Engrafted hIL-3/GM-CSF h/m KI mice were infected by intravenousinjection with Bacillus Calmette-Guérin (BCG), an attenuated strain ofM. bovis that is used as a vaccine against tuberculosis in humans. Miceused for BCG infection experiments had blood engraftment levels of >20%hCD45+ cells with >8% of hCD45+ cells being T cells (hCD3+). Mice were9-10 weeks old at the time of infection. Mice were infected with 1×10⁵colony-forming units of BCG (Statens Serum Institute Copenhagen) in avolume of 0.1 ml by tail vein injection.

Since T cells are essential for granuloma formation, first the human Tcell response to BCG four weeks after infection was examined. Flowcytometry clearly demonstrated the presence of human T cells in thelungs of both engrafted m/m and h/m mice (FIG. 9(a)). In fact, T cellswere the predominant human hematopoietic cell type in BCG-infectedlungs. Compared to m/m control mice, h/m mice infected with BCG had ahigher average ratio of human CD4 to CD8 T cells in the lungs althoughthis difference did not quite reach statistical significance (FIG. 9(b),9(c)). There was no difference in the splenic hCD4/hCD8 T cell ratiobetween the two groups of mice.

Next, the expression of two T cell-derived cytokines was analyzed,namely IFNγ and TNFα, both of which are crucial for the protectiveimmune response against mycobacteria. BCG-specific IFNγ production byintracellular cytokine staining after restimulation of splenocytes frominfected mice with peptides derived from the immunodominantmycobacterial antigen TB10.4 was examined. As expected, a population ofIFNγ-producing mouse T cells could be detected among splenocytes fromBALB/c mice (FIG. 9(d)). In addition, a human BCG-specific T cellresponse was found in a subset of engrafted h/m and m/m mice (FIG.9(d)). Finally, the majority of engrafted h/m and m/m mice expressedhuman IFNγ and TNFα mRNA in the lung after BCG infection (FIG. 9(e)).These results show that a subset of engrafted mice are capable ofmounting a pathogen-specific human T cell response to BCG, although thisresponse was not enhanced in hIL-3/GM-CSF KI mice. Consistent with this,the bacterial burden was not different between h/m and m/m mice.

Next, granuloma formation was assessed in lung and liver by histologyfour weeks post-infection. Non-engrafted m/m mice (lacking T cells) didnot develop granulomas (Table 1), which is consistent with therequirement for T cells for granuloma formation. Similarly, m/m miceengrafted with human cells did not show any granulomas in either lung orliver (Table 1). In contrast, the majority of h/m mice had small lesionsor granulomas in either lung (FIG. 10(a)) or liver or in both organs(Table 1). In general, the observed granulomas were small and had theloose organization more typical of granulomas in mice than in humans.However, lung granulomas in hIL-3/GM-CSF KI mice contained humanhematopoietic cells (hCD45+) as demonstrated by immunohistochemistry(FIG. 10(b)). The majority of these cells were human T cells (hCD3+)with a few centrally located human macrophages (hCD68+) (FIG. 10(b). Insummary, engrafted hIL-3/GM-CSF KI mice are capable of developinggranulomas that contain human T cells and human macrophages in responseto mycobacterial infection, which has not been previously reported inHIS mice. Table 1 lists lesions/granulomas found in liver and lungtissue sections from BALB/c, non-engrafted m/m (non), engrafted m/m, andengrafted hIL-3/GM-CSF h/m KI mice four weeks after BCG infection.

TABLE 1 Granulomas in BCG-Infected Engrafted hIL-3/GM-CSF KI Mouse #IL-3/GM-CSF Liver Lung 1 m/m (non) No lesions No lesions 2 m/m (non) Nolesions No lesions 3 m/m (non) No lesions No lesions 4 m/m (non) Smalllesions No lesions 1 m/m No lesions No lesions 2 m/m No lesions Nolesions 3 m/m No lesions No lesions 4 m/m No lesions No lesions 5 m/m Nolesions No lesions 6 m/m No lesions No lesions 1 h/m No lesions Nolesions 2 h/m Neutrophilic lesion Granulomas 3 h/m Small lesions Nolesions 4 h/m Small lesions No lesions 5 h/m Small lesions No lesions 6h/m No lesions Granulomas 7 h/m Granulomas No lesions 1 BALB/c Nolesions No lesions 2 BALB/c Small lesions No lesions 3 BALB/c Smalllesions No lesions 4 BALB/c Granulomas No lesions

FIG. 9(a)-(g) shows human T cell response to BCG in engraftedhIL-3/GM-CSF KI mice. FIG. 9(a)-(c) shows flow cytometry analysis oflung cells from engrafted m/m and h/m KI mice four weeks after BCGinfection. FIG. 9(a) shows frequency of human T cells (hCD45+hCD3+) inthe lung. Numbers next to boxed areas indicate percentages of cells.FIG. 9(b) shows the distribution of human CD4 and CD8 T cells in thelung. Dots plots are gated on hCD45+hCD3+ cells. Numbers in quadrantsindicate percentages of cells. FIG. 9(c) shows the ratio of human CD4 toCD8 T cells in lung (each n=6). Each dot represents one mouse.Horizontal bars indicate mean values. FIG. 9(d) shows flow cytometryanalysis of splenocytes from BALB/c mice, engrafted m/m mice, andengrafted h/m KI mice four weeks after BCG infection. Splenocytes wererestimulated in vitro with a pool of overlapping peptides covering theTB10.4 protein as described herein. Dot plots show the frequencies ofmouse IFNγ+ CD4 T cells (mCD4+) or human IFNγ+ T cells (hCD3+) asdetermined by intracellular cytokine staining. Staining withisotype-matched abs was used as a control. FIG. 9(e) shows the resultsof quantitative RT-PCR analysis of human IFNγ (left) and TNFα (right)gene expression in lung tissue from BALB/c mice, non-engrafted (non) m/mmice, engrafted m/m mice, and engrafted h/m KI mice four weeks after BCGinfection (n=4-7 per group). Each dot represents one mouse. Horizontalbars indicate mean values.

FIG. 10(a),(b) show that engrafted hIL-3/GM-CSF KI mice developgranulomas containing human cells after BCG infection. FIG. 10(a) showshematoxylin and eosin (H&E) staining of lung tissue sections fromengrafted h/m KI mice four weeks after BCG infection (magnification 100×(left) and 200× (right)). FIG. 10(b) shows immunohistochemistry of lungtissue sections stained for human CD45, CD3, or CD68 from engrafted h/mKI mice four weeks after BCG infection (magnification 200×). Onerepresentative example of two mice with lung granulomas is shown.

Example 10 hTPO Mice Engraftment and Analysis

Transplantation into TPO Mice.

Recipient mice were engrafted with human hematopoietic progenitors asdescribed in Traggiai et al. Cord blood samples were collected fromhealthy full-term newborns, under approval from the Yale humaninvestigations committee (Department of Labor and Birth, Yale New HavenHospital, New Haven, Conn.). Fetal liver samples were obtained from theHuman Fetal Tissue Repository at Albert Einstein College of Medicine,Bronx, N.Y.; and from Advance Biosciences Resources, Inc., Alameda,Calif.

Fetal liver samples were cut in small fragments, treated for 45 minutesat 37° C. with Collagenase D (100 ng/ml, Roche) and a cell suspensionwas prepared. Human CD34+ cells were purified from fetal liver samplesor from cord blood by density gradient centrifugation (LymphocyteSeparation Medium, MP Biomedicals) followed by positive immunomagneticselection using anti-human CD34 microbeads according to themanufacturer's instructions (Miltenyi Biotec). Cells were either frozenin 10% DMSO containing FBS or injected directly.

Newborn pups (within first day of life) were sublethally irradiated(X-ray irradiation, 2×150 cGy 4 hours apart) and 100,000 to 200,000CD34+ cells in 20 microliters of PBS were injected into the liver usinga 22-gauge needle (Hamilton Company, Reno, Nev.).

All experiments were performed in compliance with Yale University HumanInvestigation Committee protocol and Yale Institutional Animal Care andUse Committee protocols.

TPO Expression.

Serum concentrations of mouse and human TPO protein were measured byspecies-specific ELISA (RayBiotech) following the manufacturer'sprotocol. To measure the expression of mouse and human mRNA encodingTPO, tissues were isolated from adult animals and total RNA was purifiedusing TRIzol (Invitrogen) following the manufacturer's instructions.Contaminating genomic DNA was eliminated by treatment with RNase-FreeDNase I (Roche) and the RNA was reverse-transcribed using SuperScript IIreverse transcriptase (Invitrogen) and oligo-dT primers. The followingprimers were used for PCR amplification: mTpo forward, CCACCACCCATGGATCTC (SEQ ID NO:1); mTpo reverse, AAAGCAGAAC ATCTGGAGCA G (SEQ IDNO:2); hTPO forward, CAGGACTGAA AAGGGAATCA (SEQ ID NO:3); hTPO reverse,CGTTGGAAGG CCTTGAATTT (SEQ ID NO:4); mRpI13a forward, GTACGCTGTGAAGGCATCAA (SEQ ID NO:5); mRpI13a reverse, ATCCCATCCA ACACCTTGAG (SEQ IDNO:6).

To determine whether human TPO is faithfully expressed in these mice,total RNA was extracted from a variety of organs from a TPO^(h/m) mouse,and a similar pattern of expression for both mouse and human mRNAencoding TPO by RT-PCR was observed (FIG. 11(a)). Next, the expressionin three tissues or cell types known to express TPO (liver, kidney andmesenchymal multipotent stroma cells) from TPO^(m/m), TPO^(h/m) andTPO^(h/h) mice were compared. The expression of mouse Tpo in samplesfrom TPO^(m/m) and TPO^(h/m) mice was detected, while human TPO wasexpressed in TPO^(h/m) and TPO^(h/h) (FIG. 11(b)). The concentrations ofTPO protein in the serum of the targeted mice were also measured. MouseTPO was detected in TPO^(m/m) and TPO^(h/m) animals, and human TPO inTPO^(h/m) and TPO^(h/h) (FIG. 11(c)). The concentrations measured forhuman TPO were approximately 10-fold lower than mouse TPO. However, thisdifference is compatible with the physiological concentrations reportedin healthy human and mouse (FIG. 11(c)), and might be due tospecies-specific differences of cytokine half lives.

FIG. 11 shows (a) RT-PCR analysis of mouse TPO (m Tpo) and human TPO(hTPO) expression in different tissues of a Rag2^(+/−)γ_(c) ^(Y/−)TPO^(h/m) mouse; mouse RpI13a was used as housekeeping gene; (b) RT-PCRanalysis of m Tpo and hTPO expression in liver, kidney and mesenchymalmultipotent stromal cells (MSCs) of Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m),TPO^(h/m) and TPO^(h/h) mice; (c) concentrations of mouse and human TPOproteins measured by ELISA in serum of TPO^(m/m), TPO^(h/m) andTPO^(h/h) mice (in pg/ml, mean±S.D., n=7-9). ND: not detected; thenormal ranges indicated are from R&D Systems, Thrombopoietin Quantikinekits.

Mesenchymal Multipotent Stroma Cell Isolation.

For TPO studies, femur and tibia of mice were harvested and the bonemarrow cells were flushed out. Bones were cut into small pieces anddigested with collagenase P and D (10 μg/ml) for 45 minutes at 37° C.Bone associated cells were collected by repeated pipetting. Cells werecultured in the presence of MSC medium with stimulatory supplements(Stemcell Technologies) for 14 days. Hematopoietic lineage cells wereremoved from the culture through immunomagnetic cell sorting (MACS,Miltenyi Biotec) using CD45 and Ter119 antibodies. Non-hematopoieticcells (CD45-Teri 19-) were cultured for 5 more days and MSC phenotype(CD45-Ter119-Sca1+CD90+) was confirmed by FACS (Diminici et al. (2006)Minimal criteria for defining multipotent mesenchymal stromal cells, TheInternational Society for Cellular Therapy position statement,Cytotherapy 8:315-317).

Analysis of Hematopoietic Cell Populations in TPO Mice.

The mice were bled 8-12 weeks after transplantation. Red blood cellswere lysed three times using ACK (Lonza), and the cells were stainedwith anti-mouse CD45 and anti-human CD45 antibodies. Animals in which atleast 1% of CD45+ cells were of human origin, were used for furtheranalysis. Approximately 80% of the transplanted mice reached thisengraftment threshold, and no difference was noticed between the TPOm/mand TPOh/h groups.

The mice were sacrificed at 3-4 or 6-7 months after engraftment. Singlecell suspensions were prepared from the bone marrow (flushed from 2femurs and 2 tibias), spleen and thymus. Red blood cells were eliminatedby ACK lysis and cells were stained for FACS analysis using thefollowing antibodies. For overall hematopoietic engraftment: anti-mouseCD45-eFluor450 (30-F11, eBioscience) and anti-human CD45-APC-Cy7 (2D1).For human hematopoietic stem and progenitor cells and hematopoieticlineages: anti human CD14-PerCP (MoP9), CD19-APC (HIB19), CD33-APC(WM53), CD34-PE (AC136, Miltenyi Biotec), CD38-FITC (HIT2), CD41a-APC(HIPS) and CD66-FITC (B1.1).

For mouse stem and progenitor cell analysis, the anti-lineage cocktailcontained biotinylated antibodies against CD3ε (145-2C11), CD11b(M1/70), CD11c (HL3), CD19 (1D3), Gr1 (RB6-8C5) and Ly-76 (Ter119).Cells were subsequently stained with streptavidin-APC-Cy7, anti cKit-APC(2B8) and anti Sca1-PE-Cy7 (D7).

All the antibodies were obtained from BD Biosciences, except otherwisespecified. The data were acquired on a FACSCalibur™ or LSRII™ flowcytometer (BD Biosciences) and analyzed using the FlowJo™ software.

Functional Characterization of Human Hematopoietic Stem and ProgenitorCells in TPO Mice.

Bone marrow cells from 3 to 7 engrafted mice were pooled and human CD34+cells were purified by MACS depletion of mouse CD45+ cells (MiltenyiBiotec) followed by FACS sorting of human CD45+ CD34+ cells on aFACSAria™ flow cytometer (BD Biosciences).

To assess the colony forming capacity of human CD34+ cells, IMDM wassupplemented with 20% FCS, 2 mM L-glutamine, 55 μM 2-mercaptoethanol(all reagents from GIBCO) mixed with Methocult™ H4100, 1% BSA (StemcellTechnologies) and the following human cytokines were added: SCF (10ng/ml), FLT3I (10 ng/ml), TPO (50 ng/ml), IL-3 (20 ng/ml), IL-6 (10ng/ml), IL-11 (10 ng/ml), GM-CSF (50 ng/ml), EPO (4 U/ml) (all from R&Dsystems). 100,000 to 150,000 sorted cells were plated on 60 mm petridishes and incubated at 37° C., 5% CO₂ for 12-14 days. The number ofcolonies at 12-14 days was counted and categorized into specific myeloidlineage by microscopy.

For secondary transplantation experiments, 100,000 CD34+ cells purifiedfrom TPO^(m/m) or TPO^(h/h) primary recipients were injected intosublethally irradiated (2×200 cGy) Rag2^(−/−)γc^(−/−) TPO^(m/m)secondary recipients, as described above. These mice were sacrificed 8weeks later and the percentage of human CD45+ cells in bone marrow wasdetermined by FACS.

Statistical Analysis of TPO Mice Data.

Data were compared using two-tailed unpaired t-test. When more than 2samples were compared, one-way ANOVA followed by Tukey post hoc testswas performed. The proportions of engrafted mice in the secondarytransplantation experiment were compared using Pearson's Chi squaredtest. Differences were considered significant when the p values werelower than 0.05.

Example 11 hTPO Engrafted Mice Improved Human Engraftment Levels inTPO^(h/h) Recipient Mice Bone Marrow

Phenotyping of Bone Marrow of Humanized and Engrafted Mice.

Cells isolated from bone marrow of humanized mice were analyzed by flowcytometry and showed statistically significant improvements inengraftment of total human hematopoietic cells, human hematopoietic stemcells, human myeloid cells, human and granulocytes relative toengraftment of non-humanized mice (La, RAG and II2rg knockouts lackinghumanization of TPO gene). See FIGS. 12 & 14.

Rag2^(−/−)γ_(c) ^(−/−) mice with wild-type Tpo (TPO^(m/m)), heterozygous(TPO^(h/m)) or homozygous (TPO^(h/h)) TPO gene replacement were preparedas described. Irradiated (2×1.5 Gy) newborn Rag2^(−/−)γ_(c)^(−/−)TPO^(m/m) and TPO^(h/h) mice were engrafted with human CD34⁺ cellspurified from cord blood or fetal liver and analyzed engraftment in bonemarrow 3-4 months or 6-7 months later.

FIG. 12 shows results of engraftment studies. FIG. 12(a) showsrepresentative FACS analysis of human and mouse CD45⁺ cells in bonemarrow of Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) mice 3 to 4months after engraftment with human CD34⁺ cells. Results of tworepresentative mice are shown for each genotype. Percentages of mouseand human CD45⁺ cells among the total (mouse+human) CD45⁺ cellpopulations are indicated. FIG. 12(b) shows percentages of human CD45⁺cells in the bone marrow 3 to 4 months (left, n=42-53) or 6 to 7 months(right, n=20-25) after transplantation. Each symbol represents anindividual mouse, horizontal bars indicate mean values. FIG. 12(c) showsabsolute numbers of human CD45⁺ cells in the bone marrow of the sameanimals as in (b). P values indicate statistical significance.

A significant increase was observed in the percentages (FIGS. 12(a) and12(b)) and absolute numbers (FIG. 12(c)) of human hematopoietic cells(hCD45⁺) in bone marrow of TPO^(h/h) compared to TPO^(m/m) recipients atboth time points. Furthermore, TPO^(h/h) recipients displayed a lowerengraftment variability, with an at least 80% human chimerism in 75% ofthe mice at 3-4 months (FIG. 12(b)). The source of the CD34⁺ cells didnot affect this result, as a similar increase in chimerism in TPO^(h/h)hosts was observed with cells derived from cord blood and from fetalliver (FIG. 12(d)). Interestingly, while numbers of human cells declinedin TPO^(m/m) hosts between the early and later time points, theyremained constant in TPO^(h/h) animals (FIG. 12(c)). These results areconsistent with previously described functions of TPO in the mouse.First, TPO favors the expansion of HSCs after transplantation intoirradiated recipient mice, leading to increased engraftment levels;second, it favors the maintenance of adult HSCs, leading to sustainedhematopoiesis throughout adult life.

Example 12 hTPO Engrafted Mice Effect of TPO Humanization on Mouse andHuman Platelets

Platelet Analysis in TPO Mice.

Platelet counts in peripheral blood were measured using a Hemavet™ 950FSmachine (Drew Scientific). Blood samples were then stained withanti-mouse CD61-PE (2C9.G2) and anti-human CD41a-APC (HIPS), and thepercentages of mouse and human platelets were determined by flowcytometry, without placing any gate on the size (FSC) or granulosity(SSC) of the cells. The absolute mouse and human platelet counts werecalculated by multiplying these respective percentages with the absoluteplatelet counts.

As TPO is well known for its crucial function on thrombopoiesis, it wasinvestigated whether TPO humanization affected platelet development.Humanization of both alleles of the TPO gene led to an approximatelytwo-fold reduction in blood platelet counts of non-engraftedRag2^(−/−)γ_(c) ^(−/−) mice (FIG. 13(a)). After engraftment with humancells, the counts of mouse platelets in TPO^(h/h) mice were furtherdecreased, to less than 25% of normal values (FIG. 13(d)). The ratio ofhuman to mouse platelets (FIG. 13(b), 13(c)), as well as the absolutecounts of human platelets (FIG. 13(e)), tended to be higher in TPO^(h/h)mice than in TPO^(m/m), but none of these differences reachedstatistical significance. Furthermore, the percentage of bone marrowmegakaryocytes (CD41a⁺ cells) among human cells was comparable in bothstrains (FIG. 13(f)). These results demonstrate that levels or biologicactivity of human TPO reached by the knock-in strategy are notsufficient to fully replace mouse TPO function, and furthermore suggestthat human TPO on its own is not sufficient to support humanthrombopoiesis in the mouse environment.

FIG. 13(a) shows platelet counts in the blood of adult non-engraftedRag2^(−/−)γ_(c) ^(−/−) TPO^(m/m), TPO^(h/m) and TPO^(h/h) mice. p<0.0001(one way ANOVA, n=7-17; the indicated p-values were calculated with theTukey post hoc test). Each symbol represents an individual mouse,horizontal bars indicate mean values; (b) representative FACS analysisof mouse (mCD61⁺) and human (hCD41a⁺) platelets in the blood ofRag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) mice 3 to 4 months afterengraftment. The numbers indicate percentages among total events; (c)human platelet chimerism, determined by FACS, in TPO^(m/m) and TPO^(h/h)mice (n=19-22). Only mice with a percentage of human CD45⁺ cells in theblood higher than 5% were included in this analysis; (d),(e) counts ofmouse (mCD61⁺, (d)) and human (hCD41a⁺, (e)) platelets in the blood ofTPO^(m/m) and TPO^(h/h) recipients; (f) human megakaryocyte percentages(CD41a⁺) among human CD45⁺ cells in the bone marrow.

FIG. 13(g)-(i) show human engraftment levels in secondary lymphoidorgans. FIG. 13 (g),(h) provides percentages of human CD45⁺ cells inblood (20 g; n=43-53) and spleen (13 h; n=35-36) of Rag2^(−/−)γ_(c)^(−/−) TPO^(m/m) and TPO^(h/h) mice (each symbol represents anindividual mouse, horizontal bars indicate mean values); (i) shows totalcellularity of the thymi of engrafted TPO^(m/m) and TPO^(h/h) recipients(n=24-34). More than 90% of the cells found in the thymus were of humanorigin (hCD45⁺).

Example 13 hTPO Engrafted Mice Multi-Lineage Hematopoiesis inTPO-Humanized Mice

Phenotyping of Blood Cells of Humanized and Engrafted Mice.

Cells isolated from blood of humanized mice were analyzed by flowcytometry and showed statistically significant improvements inengraftment of human monocytes and granulocytes relative to engraftmentof non-humanized mice (i.e., RAG and II2rg knockouts lackinghumanization of TPO gene). See FIG. 14.

It was investigated whether human TPO could favor multilineagedifferentiation of human hematopoietic stem and progenitor cells invivo. As previously reported (Traggiai et al.; Ishikawa et al. (2005)Development of functional human blood and immune systems in NOD/SCID/IL2receptor gamma chain (null) mice, Blood 106:1565-1573)), the engraftedhuman cells gave rise mostly to B cells (CD19⁺) in wild-typeRag2^(−/−)γ_(c) ^(−/−) hosts (61.51±4.71% of the human cells in thespleen, mean±sem, n=32), with only a small fraction of myeloid cells.When TPO^(m/m) and TPO^(h/h) recipients were compared, a significantincrease in frequency of CD33⁺ myeloid cells in the bone marrow ofTPO^(h/h) mice was observed (FIGS. 14(a) and 14(b)). Interestingly, thisincrease was mostly due to granulocytes (CD33⁺CD66^(hi)SSC^(hi) cells),while the fraction of monocytes (CD33^(hi)CD66^(lo)CD14⁺) was similar inboth strains (FIG. 14(a),(c),(d),(e)). The percentage of myeloid cells(both granulocytes and monocytes) was also significantly increased inthe peripheral blood of TPO^(h/h) animals (FIGS. 14(a),(f), and (g)).

FIG. 14(a)-(g) depicts improved multilineage hematopoiesis in hTPO miceas measured by CD33⁺, CD66⁺, CD14⁺ cells in engrafted TPO^(m/m) andTPO^(h/h) mice. FIG. 14(a) shows representative FACS analysis of humanmyeloid cell populations in bone marrow and blood of Rag2^(−/−)γ_(c)^(−/−) TPO^(m/m) and TPO^(h/h) mice 3 to 4 months after engraftment. Thenumbers indicate the percentages among the indicated gated cellpopulations. FIGS. 14(b)-(e) show analysis of human myeloid cellpopulations relative to total human CD45⁺ cell chimerism in bone marrowof Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) recipients (n=19).FIG. 14(b) provides total myeloid populations (CD33+ cells). FIG. 14(d)provides granulocytes (CD33⁺CD66^(hi)), FIG. 14(c) shows Diff-Quick™staining of hCD45⁺SSC^(hi)CD33⁺CD66^(hi) cells purified from the bonemarrow of TPO^(h/h) recipients. FIG. 14(e) shows monocytes(CD33⁺CD66^(lo)CD14⁺). Each symbol represents an individual mouse,horizontal bars indicate mean values. FIGS. 14(f) and (g) show analysisof human myeloid cell populations relative to total human CD45⁺ cellchimersim in blood of Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h)recipients (n=6-7); FIG. 14(f): granulocytes (CD66⁺); FIG. 14(g):monocytes (CD14⁺).

Example 14 hTPO Engrafted Mice Humanization Effect on Mouse and HumanHematopoietic Stem and Progenitor Cells

The effect of human TPO on the number and function of HSCs andprogenitor cells themselves was analyzed. Genetic deletion of TPO leadsto a reduction of HSCs in adult mice. To determine whether TPOhumanization could affect the mouse population immunophenotypicallydefined as containing mouse HSCs, the percentages of lineage-negativeSca1⁺ c-Kit⁺ cells in bone marrow of non-engrafted TPO^(m/m), TPO^(h/m)and TPO^(h/h) adult mice were compared.

FIG. 15 shows decreased mouse lin⁻c-Kit⁺Sca1⁺ cells and increased numberand self-renewal potential of human stem and progenitor cells in bonemarrow of human TPO knock-in mice. FIG. 15(a) shows representativeresults of FACS analysis of mouse Lin⁻ Sca1+ c-Kit⁺ stem and progenitorcells in the bone marrow of non-engrafted Rag2^(−/−)γ_(c) ^(−/−)TPO^(h/m) and TPO^(h/h) mice compared to WT TPO(TPO^(m/m) Rag)2^(−/−)γ_(c) ^(−/−) mice. Numbers indicate the percentageof Sca1⁺ c-Kit⁺ cells among the Lin⁻ population. FIG. 15(b) showsquantitative analysis of the results presented in (a). p=0.0006 (one wayANOVA; the indicated p-values were calculated with the Tukey post hoctest; n=5/per genotype and the presented results are representative of 2independent experiments). Each symbol represents an individual mouse,horizontal bars indicate mean values. FIG. 15(c) shows representativeFACS analysis of human CD34⁺CD38⁻ cells in the bone marrow ofRag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) mice 3 to 4 months afterengraftment. The numbers indicate the percentage of CD38⁻ cells amongthe human CD45⁺CD34⁺ cells. FIG. 15(d) shows quantitative analysis ofthe percentages of CD38⁻ cells in the human CD45⁺CD34⁺ population inTPO^(m/m) and TPO^(h/h) recipient mice (n=43-53). FIG. 15(e) showsabsolute numbers of human CD34⁺CD38⁻ cells in the bone marrow of thesame mice as in 15(d)d. FIGS. 15(f) and (g) show methylcellulose colonyformation assay with human CD45⁺CD34⁺ cells purified fromRag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) and TPO^(h/h) recipients. FIG. 15(f) isCFU-GEMM, FIG. 15(g) is BFU-E (black), CFU-G (white), CFU-M (gray) andCFU-GM (dashed). CD34⁺ cells were pooled from groups of 3-4 mice, 4independent pools per genotype of recipient mice. In FIG. 15(h) humanCD45⁺CD34⁺ cells were purified from Rag2^(−/−)γ_(c) ^(−/−) TPO^(m/m) andTPO^(h/h) primary recipient mice, transplanted into newbornRag2^(−/−)γ_(c) ^(−/−) mice (100,000 cells per mouse), and human CD45⁺chimerism was determined in secondary recipients 8 weeks later. Theresults are pooled from two independent experiments (n=7-12 primaryrecipients, n=11-19 secondary recipients).

A significant reduction in the percentage of these cells in bothTPO^(h/m) and TPO^(h/h) mice compared to TPO^(m/m) as observed (FIG.15(b)), suggesting that human TPO is either not fully cross-reactive onthe mouse receptor or is not available in sufficient amounts to mousecells in this knock-in setting.

Human CD34⁺ populations in the bone marrow of engrafted TPO^(m/m) andTPO^(h/h) hosts were characterized. Human HSCs with long-termrepopulating potential are contained in the Lin-CD34⁺CD38⁻ cellfraction. The percentage of CD34⁺ cells among the human CD45⁺ populationwas slightly increased in TPO^(h/h) mice (12.39±0.79% vs. 10.00±0.81%,mean±sem, n=43-53, p=0.037). A small (1.5 fold) but statisticallysignificant increase was observed in the percentage of CD38⁻ cellswithin the CD34⁺ population in TPO^(h/h) compared to TPO^(m/m)recipients (FIG. 15(c),(d)). Overall, this resulted in a significantincrease (approximately 2.8-fold) of absolute numbers of CD34⁺CD38⁻cells in TPO-humanized mice (FIG. 15(e)). Thus, based on cell surfaceimmunophenotype, human TPO favors a population of cells known to behighly enriched for HSCs.

To address the functional properties of this cell population, humanCD34⁺ cells were purified from the bone marrow of TPO^(m/m) andTPO^(h/h) mice, and assessed in methylcellulose colony formation assaysin vitro. CFU-GEMM are multilineage myeloid colonies derived fromimmature cells that at least contain all erythro-megakaryocyte andmyeloid cell differentiation potential. The formation of CFU-GEMM wasdetected, albeit in small numbers, from all four samples of CD34⁺ cellsisolated from TPO^(h/h) recipient mice, while only one sample fromTPO^(m/m) generated CFU-GEMM (FIG. 15(f)). This result demonstratesimproved maintenance of immature human hematopoietic progenitor cells inTPO^(h/h) recipients. Furthermore, consistent with enhanced myeloiddifferentiation observed in vivo (FIG. 14), the numbers of CFU-M werealso significantly higher in human CD34⁺ cell samples isolated fromTPO^(h/h) compared to TPO^(m/m) mice (225.0±12.25 vs. 81.25±10.80colonies per 150,000 CD34⁺ cells plated, mean±sem, p=0.0001; FIG.15(g)).

Maintenance and/or self-renewal of HSCs is best demonstratedfunctionally by successful secondary transplants. SCID repopulatingcells (SRCs) that serially engraft in mice represent currently thesurrogate experimental gold standard for human HSC function. Thus, humanCD34⁺ cells were purified from bone marrow of TPO^(m/m) and TPO^(h/h)primary recipients and transplanted in equally low numbers (100,000CD34⁺ cells per animal) into Rag2^(−/−)γ_(c) ^(−/−) newborn mice. Bonemarrow of secondary recipients was analyzed 8 weeks later (FIG. 15(h)).Human CD34⁺ cells isolated from TPO^(m/m) primary recipients had a verylow capacity to serially engraft, as human CD45⁺ cells were detected inonly 2 of 11 secondary recipients. By contrast, human CD45⁺ cells werepresent in the bone marrow of 15 of 19 mice engrafted with CD34⁺ cellsisolated from TPO^(h/h) primary recipients (p=0.0012). As the genotypeof the secondary recipient mice was the same for both groups(TPO^(m/m)), this result indicates that the presence of human TPO in theprimary recipient favored the maintenance of human cells with enhancedself-renewal capacity.

Taken together, these results demonstrate that homozygous TPO-humanizedmice represent a better environment to maintain self-renewal capacityand multilineage differentiation potential of human hematopoietic stemand progenitor cells.

Persons skilled in the art can devise various arrangements that,although not explicitly described or shown in this disclosure, embodythe invention and are included within its spirit and scope. All examplesare provided to help the reader understand the principles and conceptsof the invention and are used without limitation to the specificexamples and embodiments described. All principles, aspects,embodiments, and examples of the invention are intended to encompassequivalents thereof, whether the equivalents are now known or developedin the future. The scope of the present invention is not intended to belimited to the embodiments and examples shown and described in thisdisclosure.

We claim:
 1. A genetically modified mouse, comprising a replacement of amouse thrombopoietin (TPO) gene with a human TPO gene at both alleles ofa mouse TPO gene locus, wherein the mouse does not express mouse TPO,wherein the mouse is immunocompromised for a mouse immune system,wherein the mouse is engrafted with human hematopoietic cells, andwherein the mouse comprises a statistically significant increase inhuman CD45+ hematopoietic cells in the bone marrow relative to a humanhematopoietic cell-engrafted mouse expressing mouse TPO.
 2. The mouseaccording to claim 1, wherein the mouse comprises a humanhemato-lymphoid system.
 3. The mouse according to claim 2, wherein thehuman hemato-lymphoid system comprises human cells selected from thegroup consisting of hematopoietic stem cells, hematopoietic CD34+ cells,myeloid precursor cells, myeloid cells, dendritic cells, monocytes,granulocytes, neutrophils, mast cells, lymphocytes, and platelets. 4.The mouse according to claim 2, wherein the mouse is null for a RAG geneand null for the mouse interleukin 2 receptor gamma (IL-2Rγ) gene.
 5. Amethod of producing a mouse comprising a human hemato-lymphoid system,the method comprising: engrafting a population of cells that compriseshuman hematopoietic cells into a immunodeficient genetically modifiedmouse, wherein the genetically modified mouse comprises a replacement ofa mouse thrombopoietin (TPO) gene with a human TPO gene at both allelesof a mouse TPO gene locus, wherein the mouse does not express mouse TPO,and wherein the engrafted mouse comprises a statistically significantincrease in human CD45+ hematopoietic cells in the bone marrow relativeto a human hematopoietic cell-engrafted mouse expressing mouse TPO. 6.The method according to claim 5, wherein said population of cellscomprises a population of human umbilical cord blood cells or humanfetal liver cells.
 7. The method according to claim 5, wherein saidpopulation of cells comprises human CD34+ cells.
 8. The method accordingto claim 5, wherein the human hemato-lymphoid system comprises humancells selected from the group consisting of hematopoietic stem cells,myeloid precursor cells, myeloid cells, dendritic cells, monocytes,granulocytes, neutrophils, mast cells, lymphocytes, and platelets. 9.The method according to claim 5, further comprising: irradiating thegenetically modified mouse prior to the engrafting.
 10. The methodaccording to claim 5, wherein the mouse is null for a RAG gene and nullfor the mouse interleukin 2 receptor gamma (IL-2Rγ) gene.
 11. The methodaccording to claim 5, further comprising assessing the humanhemato-lymphoid system for human hematopoietic cells, wherein the humanhemato-lymphoid system comprises an enhanced percent of humanhematopoietic cells relative to percent of human hematopoietic cells inan engrafted mouse that lacks a humanization of a TPO gene.
 12. Themethod according to claim 11, wherein the assessing comprises detectingmyeloid cells, wherein the human hemato-lymphoid system comprises anenhanced percent of myeloid cells relative to percent of myeloid cellsin an engrafted mouse that lacks a human TPO gene.
 13. The methodaccording to claim 11, wherein the assessing comprises detectinggranulocytes, wherein the human hemato-lymphoid system comprises anenhanced percent of granulocytes relative to percent of granulocytes inan engrafted mouse that lacks a human TPO gene.
 14. The method accordingto claim 11, wherein the assessing comprises detecting hematopoieticstem cells, wherein the population comprises an enhanced percent ofhematopoietic stem cells relative to percent of hematopoietic stem cellsin an engrafted mouse that lacks a human TPO gene.
 15. A mousecomprising a humanized cellular immune system, wherein the mouse is nullfor a RAG gene and null for the mouse interleukin 2 receptor gamma(IL-2Rγ) gene and comprises: a replacement of a mouse thrombopoietin(TPO) gene with a human TPO gene at both alleles of a mouse TPO genelocus, wherein the mouse does not express mouse TPO; and a humanhemato-lymphoid system, wherein the mouse comprises a statisticallysignificant increase in human CD45+ hematopoietic cells in the bonemarrow relative to a mouse comprising a human hemato-lymphoid system andexpressing mouse TPO.
 16. The mouse according to claim 15, wherein themouse comprises a S. typhi infection.
 17. The mouse according to claim16, wherein the S. typhi infection is a systemic S. typhi infection. 18.The mouse according to claim 15, comprising a Mycobacterium tuberculosisinfection.